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