93773 - Clinical Analytical Chemistry

Learning outcomes

Upon completion of the course, the student possesses basic knowledge related to quantitative chemical analysis methods and the most common analysis techniques based on biospecific reagents (enzymatic analysis methods, immunometric methods). In particular, the student is able to: - solve a clinical problem in the laboratory by choosing and using one or more of the methods seen; - critically evaluate the analytical data obtained to solve the clinical problem.

Course contents

ANALYTICAL CHEMISTRY-GENERALITIES
The analytical process (common steps of chemical analysis)
Sampling of biological fluids

STATISTICS
Classes of experimental errors
Origin of experimental errors
Correction of systematic error
Accuracy and precision (definition and useful parameters to evaluate them)
Statistical parameters for processing replicate data (s, CV%, absolute E, Er%, s of the mean)
Parametric tests of significance: Student t test, Q test, F test
Confidence interval
Linear regression: Method of least squares
Calibration with external standard, internal standard and method of standard additions
Quality assessment of an analytical method: accuracy, precision (repeatability, reproducibility), sensitivity, limit of detection and quantification, dynamic range of linearity, specificity, selectivity, robustness, robustness.

SPECTROPHOTOMETRY
Lambert-Beer's law: definitions and use for quantitative analysis
Absorbance and transmittance
Absorbance measurements: choice of wavelength, deviation from linearity
Instrumentation for spectrophotometry: sources, wavelength selectors, sample holders, detectors, materials for optics
Single and dual beam spectrophotometers

CHROMATOGRAPHY
Basic concepts of chromatography: chromatogram, chromatographic peak, retention time, dead time.
Optimization of a chromatographic separation: capacity factor, selectivity, efficiency, resolution.
Chromatography instrumentation
Gas chromatography: instrumentation and methods to optimize separation.
High-performance liquid chromatography (HPLC). Instrumentation (columns, injectors, detectors).
Classes of liquid chromatography (adsorption, partitioning, ion exchange, size exclusion.
LC breakdown: normal phase and reversed phase.
Choice of mobile phase in HPLC.
Quantitative analysis in chromatography.

MASS SPECTROMETRY.
Principles and instrumental setup of mass spectrometry.
High and low fragmentation ion sources
Mass analyzers
Protein mass spectrometry

IMMUNOMETRIC METHODS
Principles and advantages of immunometric methods
Polyclonal and monoclonal antibodies. Characteristics and analytical applications.
Tracers for immunometric methods. Enzyme immunoassay methods.
Classification of immunometric methods.
Homogeneous and heterogeneous, competitive and noncompetitive methods.
Calibration curves for immunometric methods.
Quantitative parameters

ENZYMATIC METHODS
Enzymatic analysis. Principles and applications.
Properties and structure of enzymes.
Enzyme activity. Definition and units of measurement.
Enzyme kinetics.
Regions of the velocity/[substrate] curve.
Michaelis-Menten equation
Determination of enzyme activity.
One-point, two-point, multipoint methods.
Determination of analyte concentration.
Simple and coupled end-point methods.
Measurement methods in enzyme analysis.

LABORATORY EXERCISES.
Determination of active ingredient content in a pharmaceutical formulation by HPLC-UV
Quantitative analysis of protein by Bradford's method
Determination of glucose by Trinder's method.

Readings/Bibliography

- Chimica analitica clinica, D' Ovidio - De Grazia (EdiSES)

-A.J. Ninfa, D.P. Ballou "Metodologie di Base per la Biochimica e la Biotecnologia", Zanichelli (consultazione)

-J.M. Berg, J.L. Tymoczko, L. Stryer "Biochimica", V Edizione, Zanichelli

Teaching methods

Classroom lectures with projection of lecture material in electronic format that is made available on the web at http://virtuale.unibo.it

Single-place laboratory exercises with tutor support. Tutorial guides to the exercises are made available at http://virtuale.unibo.it

The teaching consists of 6 CFUs, including 4 CFUs of face-to-face lectures (32 hours) 1 CFU of classroom exercises (12 hours) and 1 CFU of laboratory exercises (15 hours). During the face-to-face lectures in which the course topics will be presented and discussed, with theoretical insights and explanatory examples. The laboratory exercises are intended to enable each student to acquire the necessary manual dexterity and knowledge of basic analytical techniques to be able to operate a laboratory according to principles of quality and safety. Classroom exercises will focus on the application of methods of statistical processing of experimental data obtained in the laboratory. The student will process the results obtained in the laboratory and submit a written report on the laboratory exercises.

In view of the types of activities and teaching methods adopted, the attendance of this training activity requires the performance by all students (including all incoming international students, e.g., ERASMUS) of modules 1 and 2 in e-learning mode [https://www.unibo.it/it/servizi-e-opportunita/salute-e-assistenza/salute-e-sicurezza/sicurezza-e-salute-nei-luoghi-di-studio-e-tirocinio] and participation in module 3 of specific training on safety and health in the workplace. Directions on dates and mode of attendance of module 3 can be found in the appropriate section of the course website ("study"--"mandatory safety and health training").

Assessment methods

Final written exam on all course topics. A detailed syllabus of the topics covered in the test will be made available at the end of class at http://virtuale.unibo.it

The test lasts 90 minutes and consists of three open-ended questions on the same number of course topics.

The evaluation criterion is based on the student's demonstration of the ability to use and apply the knowledge, information and cultural tools provided in the course. The threshold for sufficiency of learning is specifically the demonstration of critical understanding of the course topics. The lecturer is available for further clarification and to check the level of pre-exam preparation.

Evaluation of lab reports will contribute to the final grade. Reports are due at least two weeks in advance of the date on which the final exam is to be taken.

Teaching tools

Video projector, PC, labs with individual stations, equipment and glassware, spectrophotometers, plate reader, HPLC-UV.

Lecture materials: lecture materials presented in class will be made available to the student in electronic format via the Internet.

To obtain lecture materials: http://virtuale.unibo.it/

Access restricted to students enrolled in the course.

Office hours

See the website of Andrea Zattoni

See the website of Valentina Marassi