Ateneo - UPO

1_Biomarkers in laboratory medicine. The different classes of biomarkers and biological significance. Scope of use and clinical significance of biomarkers. Hints of precision and personalized medicine. How pre-analytical, analytical and port-analytical phases affect the significance of the laboratory result. The informative value of a test in the various diagnostic contexts
2_Genetic biomarkers. The Human Genome Project (HGP). Clinical applications of molecular investigations. Nucleic acid analysis techniques
3_Biochemical techniques in lab medicine
4_Protein and enzyme biomarkers. Plasma proteins and proteome. Proteomic analysis. Selection criteria and methods for determining enzymatic biomarkers in the clinical laboratory.
5_Metabolic biomarkers. Clinical applications of metabolomics analysis: congenital alterations of metabolism.

Molecular biology. Principles and techniques of Michael M. Cox et al Zanichelli.
Material provided by the teacher

Understand how metabolic, protein and genetic markers can be used in diagnostic, predictive and therapeutic settings. Acquire awareness of the limits and clinical potential of new omics technologies.
Understanding how biochemical and molecular biology techniques are used in the diagnostic field and knowing how to evaluate how the choice of an analysis method and the way in which it will be used will affect the quality of the result and the informative value it will bring to clinical practice.
Know the scope of use of enzymatic biomarkers of diagnostic interest and metabolic markers in the evaluation of organ function and in the diagnosis of metabolic disorders.

Basics of biochemistry, molecular and cell biology

Frontal lessons with slides presentation. Exposition of cases to show precise molecular diagnostic problems and guide the student to their resolution.
For the preparation of the exam students can use the material provided by the teacher (a pdf copy of the slides projected in class, of handouts prepared by the teacher and of some articles taken from scientific journals on the topics discussed during the course) and on the recommended textbook

A pdf copy of the projected slides, the in-depth material and all the information regarding the course and the methods of examination will be made available on the moodle DIR (https://www.dir.uniupo.it/)

The aim of the exam consists in verifying the level of knowledge skills developed by the student concerning the course program topics. The exam will consist in a written test including 40 questions related to all the topics of the course program divided into: quizzes with multiple choice answers or true / false and open question that require the resolution of problems concerning the determination of characteristics of a laboratory test (sensitivity, specificity and predictive values) or a short discussion of specific arguments treated during the course. The evaluation is expressed in thirtieths (minimum mark 18). During the written test it is not allowed to consult any kind of material. The calculator is allowed. The exam allows to evaluate the theoretical knowledge, the critical sense, the ability to understand the text (exercises with a request to make a choice between different alternatives) and the ability to apply the acquired knowledge to practical circumstances (numerical exercises).
The syllabus and exam modality will be available on the moodle DIR platform

1_Biomarkers in laboratory medicine
- The different classes of biomarkers and their biological significance: proteins and proteome, metabolites and metabolome, genetics and genome.
- Scope of use and clinical significance of biomarkers: diagnostic, predictive, prognostic, efficacy, evolution biomarkers.
- Notes on the meaning of precision and personalized medicine. Definition of big data, machine learning, artificial intelligence and data science.
- Interpretation and significance of the laboratory result. How the preanalytical, analytical and port-analytical phases affect the significance of the laboratory result. True variability and induced variability. Anticoagulants: mode of action. The parameters that characterize an analytical method: exactness, accuracy, precision, specificity and analytical sensitivity; robustness, measuring range, resolution, limit of detection and limit of quantification. Diagnostic sensitivity and specificity. Predictive value of a diagnostic test. The significance of the information value of a test in various diagnostic contexts
2_Genetic biomarkers
Introduction
- The Human Genome Project (HGP). History of the HGP, characteristics of the GRCh38 consensus genome. The limits of the CRCh38. Assembly of a diploid genotype. The Telomere-to-Telomere Consortium, the ENCODE Project, the Human Pangenome Project.
- Non-coding RNAs: general classification and potential clinical role
- Classification and functional consequences of genetic alterations investigated in molecular diagnostics. Clinical applications of molecular investigations.
- Potential and limitations of molecular tests. The choice of the starting biological material, according to the type of investigation and the purpose of the molecular test to be carried out.
Nucleic acid analysis techniques
- The extraction of nucleic acids from nucleated cells of peripheral blood and bone marrow and from fresh or fixed tissues. Methods of purification and quantification of nucleic acids.
- Qualitative and quantitative real-time PCR: principles and application examples for the determination of small deletions, amplifications, duplications, translocations. Examples of application of qualitative and quantitative PCR for the diagnosis and monitoring of minimal residual disease in oncology.
- Sequencing of nucleic acids. From sequencing using the Sanger technique to "single molecule sequencing".
- Analysis of structural alterations of the genome: Comparative Genomic Hybridization (CGH) array and molecular karyotype. Fluorescent in situ Hybridization (FISH).
3_Main biochemical investigation techniques
- Spectrometric analyses: general characteristics. Spectrophotometry: chromophores and chromogenic reactions. Lambert-Beer law. Kinetic reactions and end-points for the analysis of plasma metabolites and enzymes.
- The fluorescence. Fluorescence microscopy for the study of cellular components. Flow cytometry analysis of apoptosis and cell cycle.
- Electrophoretic and immunometric techniques for the study of proteins. Non-denaturing electrophoresis and denaturing electrophoresis (SDS-PAGE/western blot). Immunoprecipitation-western blot. ELISA techniques
- Chromatography and basics of mass spectrometry
4_Protein and enzyme biomarkers
Protein biomarkers in laboratory medicine
- Plasma proteins: functional classification, causes that determine their quantitative variations. Some examples relating to the determination of plasma proteins.
- Proteomic analysis with mass spectrometry. "Bottom-Up" and "Top-down" approach. Plasma proteome analysis. The “PeptideAtlas” project Other types of proteome analysis: Somascan technology and Proximity Extension Assays (PEA).
Enzymatic biomarkers
- Choice criteria for enzymatic biomarkers in the clinical laboratory. Methods for determining enzymatic activities. General factors that condition the levels of various enzymes in the blood in health and disease conditions. Factors affecting the diagnostic utility of an enzyme
- Main enzymes used in the clinical laboratory: biochemical basis of their function under normal conditions and biological rationale for their use as disease markers. Definition and diagnostic value of isoenzymes. Some examples relating to the clinical significance of the measurement of some plasma enzymes: transaminases, creatine kinase, lactate dehydrogenase.
5_Metabolic biomarkers: clinical applications of metabolomics analysis
Basics of metabolomics analysis
- Definition of metabolite, metabolome and metabolomic analysis. Definition of "Targeted" and "Untargeted" metabolomic analysis. Limitations of the "Untargeted" metabolomics analysis. Metabolomic investigation techniques: gas chromatography and HPL coupled to mass spectrometry.
Application of metabolomics in the diagnosis of congenital alterations of metabolism
- "Biochemical" classification of laboratory markers used in the diagnosis of congenital alterations of metabolism. Areas of application and limitations of proteome investigations and enzyme assays
- Investigations of the metabolome: diagnostic significance of the profile of free fatty acids, organic acids and amino acids. Examples of inborn errors of metabolism: phenylketonuria, Medium-Chain Acyl-CoA dehydrogenase (MCAD) deficiency.

Being able to autonomously evaluate how pre-analytical and analytical conditions contribute to determining the quality and informative value of laboratory results. Knowing how to evaluate the adequacy of a laboratory test according to the test chemical-physical principles and marker characteristics. Know the criteria according to which genetic, protein or metabolic biomarkers are considered most suitable in laboratory diagnostics. Acquire awareness of the clinical potential, but also the critical issues associated with untargeted omics analyses