An Introduction to Chemical Pathology 2025 Lecture PDF

An Introduction to some of the principles of Chemical Pathology LOM Feb 2025 George van der Watt, Chemical Pathologist, Red Cross Childrens Hospital, UCT and NHLS Biochemistry is the molecular basis of all life Biochemistry aims to describe, in molecular terms, all the chemical...

An Introduction to some of the principles of Chemical Pathology LOM Feb 2025 George van der Watt, Chemical Pathologist, Red Cross Childrens Hospital, UCT and NHLS Biochemistry is the molecular basis of all life Biochemistry aims to describe, in molecular terms, all the chemical processes that occur in living organisms. Chemical Pathology aims to describe , in molecular terms the derangement of normal chemical processes and how these are related to disease. For a Chemical Pathologist, every spontaneous endogenous disease has at its root a biochemical basis and every one of these diseases will eventually be unravelled and described in logical biochemical terms – this is what makes our job challenging and always interesting because we learn new things all the time. The role of a clinical chemistry laboratory is to provide accurate and relevant biochemical information for the diagnosis management of disease and to guide clinical decision making. Screening = PSA for prostate CA Diagnosis = Troponin T for a myocardial infarction Monitoring = HBA1c in a diabetic What do we measure – ALL the components of the central dogma of life Genom Transcriptom Proteom e e e Metabolom e  Measurement by proxy Chem path? Anatomical Pathology Microbiology Haematology  Using physical characteristics to elicit and measure small molecules - light All conjugated organic compounds absorb UV (190 – 400nm) or visible (400 – 700nm) light Extensively conjugated compounds absorb in the visible spectrum – eg Haemoglobin Less conjugated compounds absorb in the UV spectrum – eg many drugs and toxins UV or visible light can also excite electrons into higher energy orbits around atoms As these electrons fall back they emit light at a lower energy (longer wavelength) = fluorescence  Colorimetric assays are used to measure many small molecules, we also use them to measure enzyme velocity Enzymes are usually expressed as IU Common examples of colorimetric assays Ca, Mg, Phos ALT, AST, ALP, GGT 1 IU/L means there is enough enzyme present in 1 L of plasma to convert Albumin, Total Protein 1umol of substrate to product per minute Bilirubin Cholesterol NADH absorbs light at 340nm so we can Glucose measure LDH by measuring the Ammonia speed/rate at which absorbance Lactate decreases at 340nm when plasma is Paracetamol added to the reaction mix ION SENSITIVE ELECTRODES - POTENTIOMETRY Voltage, of an electrochemical cell is measured. The cell consists of both an indicator and reference electrode. The potential of the reference electrode is constant. The potential difference (voltage) developed at the indicator electrode contains information about the amount of analyte in a sample. During the measurement, there is little to no current flow. The key to potentiometric measurement is an ion-selective electrode (ISE) that has a high affinity membrane for a specific electrolyte and only allows that electrolyte to come close and concentrate around the electrode Many electrolytes are measured this way Na, K, Cl, ionized calcium So is pH using a special silica glass  Antibodies – for measuring low abundance molecules in immunoassays Great for bigger molecules (proteins) Non-competitive that have enough space for 2 Immunoassay antibodies to bind Here the signal is TSH, FSH, LH, PSA, Prolactin, Insulin, Proportionate to PTH – some are expressed as U/L the signal (old assays, others as mass units (mg/L) Labels use technologies that are able to amplify a small signal in a detectable manner Eg fluorophores, chemiluminescent molecules that emit light in a chemical reaction, radio-active labels, and even enzymes Used for smaller molecules Competitive that don’t have enough space Immunoassay for 2 ABs to bind but that themselves can be labelled. T4, T3, Vit D, Cortisol, Testosterone Oestrogen Here the signal is inversely Proportionate to the signal  Chromatography followed by Mass Spectrometry is the highest analytical standard – Chromatography = separation by variable affinity of molecules that are dissolved in a mobile phase for a specific stationary phase Gas Chromatography – mobile phase is helium gas, stationary phase is a melted wax th lines the inside of a very thin (30uM diameter) and long (10-30m) capillary column Many diff types of Chromatogra detectors, mass m spec is just one of them Detector High pressure/performance liquid Chromatography – mobile phase is a liquid, often a buffered aqueous solution, stationary phase = packed particles with different affinities for the analytes being separated, very high pressure is needed to push the mobile phase through these columns Mass Spectrometry There are many different forms of Mass Spec but they are all based on the following principles 1) Molecules entering the MS must be ionized (every MS has an ion source that ionizes molecules) 2) The charged molecules are accelerated in a magnetic field through a vacuum towards a detector 3) On route to the detector the molecules are separated and characterized by their mass/charge ratios (m/z) 4) Under a given set of conditions the same molecule will always behave the same in a specific instrument giving rise to a highly specific mass spectrum for each individual molecule. If we use stable isotopes as Mass Spectrum internal Is highly standards MS molecule specific becomes even = fingerprint more sensitive