Bien 545 - Ch 1 - Copy Medical Diagnosis PDF

Chapter 1 Review on medical Diagnosis Outline  Biomarkers  Image-based analysis  Cellular and chemical analysis 1 Chapter 1 Biomarkers Definition: Any substance, structure, or process that can be measured i...

Chapter 1 Review on medical Diagnosis Outline  Biomarkers  Image-based analysis  Cellular and chemical analysis 1 Chapter 1 Biomarkers Definition: Any substance, structure, or process that can be measured in the body or its products and influence or predict the incidence of outcome or disease  Biomarkers are objective medical signs (as opposed to symptoms reported by the patient) used to measure the presence or progress of disease, or the effects of treatment. Personalized medicine & treatment Early-stage diagnosis Monitoring of biomarkers 2 Chapter 1 Biomarkers Types of Biomarkers  Molecular: have biophysical properties, which allow their measurements in biological samples (eg, plasma, serum, cerebrospinal fluid, bronchoalveolar lavage, biopsy), Ex: Blood glucose  Radiographic: obtained from imaging studies, Ex: Bone mineral density  Histologic: reflect biochemical or molecular alteration in cells, tissues or fluids Ex: Grading and staging of cancers  Physiologic: measurements of body processes, Ex: Blood pressure Radiographic biomarkers Molecular biomarkers Image-based analysis cellular and chemical analysis 3 Chapter 1 Physical and image-based analysis  Diagnostic imaging describes a variety of non-invasive methods of looking inside the body to help determine the cause of an injury or an illness. Medical imaging techniques can produce pictures of the activities and structures inside your body. The main types of imaging used in modern medicine  X-ray radiography  Magnetic Resonance Imaging (MRI)  Computed Tomography (CT scanning)  Ultrasound  Nuclear medicine 4 Chapter 1 Physical and image-based analysis X-ray radiography Projectional radiography  An x-ray machine beams high-energy waves onto the body. The soft tissues, such as skin and organs, do not absorb these waves, whereas hard tissue like bones do absorb the waves. The machine transfers the results of the x-ray onto a film, showing the parts of the body that absorbed the waves (the bones) in white and leaving the unabsorbed materials in black. X-ray detectors are devices used to measure the flux, spatial distribution, spectrum, and/or other properties of X-rays. 5 https://www.youtube.com/watch?v=uPoD0jv93Ks 6 Chapter 1 Physical and image-based analysis Computed tomography (CT scanning)  In CT, a beam of X-rays spins around an object being examined and is picked up by sensitive radiation detectors after having penetrated the object from multiple angles. A computer then analyses the information received from the scanner's detectors and constructs a detailed image of the object 7 https://www.youtube.com/watch?v=l9swbAtRRbg 8 Chapter 1 Physical and image-based analysis Magnetic resonance imaging (MRI)  MRIs employ powerful magnets which produce a strong magnetic field that forces protons in the body to align with that field. When a radiofrequency current is then pulsed through the patient, the protons are stimulated, and spin out of equilibrium, straining against the pull of the magnetic field. When the radiofrequency field is turned off, the MRI sensors are able to detect the energy released as the protons realign with the magnetic field. The time it takes for the protons to realign with the magnetic field, as well as the amount of energy released, changes depending on the environment and the chemical nature of the molecules. Physicians are able to tell the difference between various types of tissues based on these magnetic properties. 9 https://www.youtube.com/watch?v=1CGzk-nV06g 10 Chapter 1 Physical and image-based analysis Ultrasound  Ultrasound waves are produced by a transducer, which can both emit ultrasound waves, as well as detect the ultrasound echoes reflected back. In most cases, the active elements in ultrasound transducers are made of special ceramic crystal materials called piezoelectrics. These materials are able to produce sound waves when an electric field is applied to them, but can also work in reverse, producing an electric field when a sound wave hits them. When used in an ultrasound scanner, the transducer sends out a beam of sound waves into the body. The sound waves are reflected back to the transducer by boundaries between tissues in the path of the beam (e.g. the boundary between fluid and soft tissue or tissue and bone). When these echoes hit the transducer, they generate electrical signals that are sent to the ultrasound scanner. Using the speed of sound and the time of each echo’s return, the scanner calculates the distance from the transducer to the tissue boundary. 11 https://www.youtube.com/watch?v=I1Bdp2tMFsY 12 Chapter 1 Physical and image-based analysis Nuclear medicine  Nuclear medicine imaging is a method of producing images by detecting radiation from different parts of the body after a radioactive tracer is given to the patient. The images are digitally generated on a computer and transferred to a nuclear medicine physician, who interprets the images to make a diagnosis. 13 Chapter 1 Physical and image-based analysis Each technique is used in different circumstances. For example,  Radiography is often used when we want images of bone structures to look for breakages.  MRI scanners are often used to take images of the brain or other internal tissues, particularly when high-resolution images are needed.  Nuclear medicine is used when you need to look inside the digestive or circulatory systems, such as to look for blockages.  Ultrasound is used to look at fetuses in the womb and to take images of internal organs when high resolution is not necessary. 14 Chapter 1 Cellular and chemical analysis  Molecular biomarkers are molecules that indicate normal or abnormal process taking place in your body and may be a sign of an underlying condition or diseases. Types of molecular Biomarkers  Small Molecules (e.g., Vitamins, Dietary Minerals)  Nucleic Acids (e.g., DNA/RNA)  Proteins Such as (e.g., inflammatory biomarkers such as cytokines) Main Sources of molecular biomarkers  Blood  urine  stool  Saliva 15 Chapter 1 Cellular and chemical analysis Gold-standard methods for analysis of molecular biomarkers Immunoassay techniques  An immunoassay is based on the selective binding between an antibody and its antigen (analyte).  Enzyme-linked Immunosorbent Assay (ELISA) is the most popular type of immunoassay techniques Polymerase chain reaction (PCR)  PCR is a method widely used to rapidly make millions to billions of copies of the target DNA/RNA region, which is initially very rare, providing various applications such as medical diagnosis. 16 Chapter 1 Cellular and chemical analysis Antibody: A protein made by plasma cells (a type of white blood cell) in response to an antigen (a substance that causes the body to make a specific immune response). Each antibody can bind to only one specific antigen.  An antibody binds to a specific region on an antigen called an epitope. A single antigen can have multiple epitopes for different, specific antibodies. 17 Chapter 1  The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen.  Determination of antigen concentration in a sample requires production of a standard curve using antigens of a known. The Sandwich ELISA concentration of antigen in a sample can then be calculated using the optical density (OD). 18  Another major benefit  sandwich ELISA is 2-5 times sensitive is that the primary method than indirect ELISA. antibody has more binding sites for the secondary antibodies  The major benefit of the indirect than the antigen has ELISA is the flexibility and choice for an antibody. This available in labeled secondary means that there is no antibodies and enzyme- problem with loss of substrate detection systems. A reactivity, and it is secondary antibody can be used actually possible to as long as it matches the host amplify the signal. species of the primary antibody. This increases the options and Indirect ELISA means that there is a high chance that an appropriate primary-secondary antibody combination will be on hand in the laboratory at all times. Also, different enzyme-substrate detection systems can be used with the same primary antibody. This allows for a greater ability to fine-tune the ELISA protocol. 19 Chapter 1 Direct ELISA  While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).  The most obvious benefit of the direct ELISA as compared to the indirect ELISA is that only one antibody is needed and it requires less time to complete the assay. The second benefit is that the potential for non-specific cross-reactivity of the secondary antibody is completely eliminated. 20 21 Chapter 1 Cellular and chemical analysis PCR  Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way.  PCR is a three-step process that is carried out in repeated cycles. The initial step is the denaturation, or separation of the two strands of the DNA molecule. This is accomplished by heating the starting material to temperatures of about 95 °C (203 °F). Each strand is a template on which a new strand is built. In the second step the temperature is reduced to about 55 °C (131 °F) so that the primers can anneal to the template. In the third step the temperature is raised to about 72 °C (162 °F), and the DNA polymerase begins adding nucleotides onto the ends of the annealed primers. At the end of the cycle, which lasts about five minutes, the temperature is raised and the process begins again. The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA. 22 Cellular and chemical analysis Schematic diagram of PCR 23 Chapter 1 Cellular and chemical analysis Quantitative/real-time PCR  Real-time PCR monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its end, as in conventional PCR, where the PCR products are visualized on agarose gel (gel electrophoresis) to determine their size as well as relative quantity  Real-time detection of PCR products is enabled by the inclusion of a fluorescent reporter molecule in each reaction well that yields increased fluorescence with an increasing amount of product DNA. The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle.  Quantitative PCR (qPCR) is used to detect, characterize and quantify nucleic acids for numerous applications. Commonly, in Reverse transcription qPCR, RNA transcripts are quantified by reverse transcribing them into cDNA first, and qPCR is subsequently carried out. As in standard PCR, DNA is amplified by 3 repeating steps: denaturation, annealing and elongation. However, in qPCR, fluorescent labeling enables the collection of data as PCR progresses. Quantitative Conventional (End-point ) 24 25 Chapter 1 Cellular and chemical analysis Clinical Significance of ELISA and qPCR as gold standard methods ELISA  Detect and measure the presence of antibodies in the blood e.g., Antibodies against infectious disease, hepatitis A, B, C, HIV, etc.  Detect and estimate the levels of tumor markers e.g., prostate-specific antigen, Carcinoembryonic Antigen (CEA)  Detect and Estimate Hormone Levels e.g., pregnancy hormone qPCR  Clinical microbiology: detection and quantification of bacterial and fungal pathogens; viral titration  oncology and Gene expression: e,g., Cancers and neurodegenerative diseases 26 Chapter 1 Limitations of gold-standard diagnostic techniques Although the gold-standard diagnostic approaches may exhibit high sensitivity and specificity, but they suffer from shortcomings such as  complicated operation procedures  labor-intensive protocols  longtime off-line assay processes  expensive equipment The mentioned issues may hinder the wide applications of these methods in clinical testing, especially in resource-poor settings. To address these challenges rapid tests and point of care diagnostic approaches have been proposed, which will be elaborated in the following chapters 27