Lap Technques Coursework Materials

LAP TECHNQUES Diagnosis vs. screening • • Diagnosis: testing to confirm whether a condition is present o Only patients with clinical signs and symptoms and a suspected condition will have this testing Screening: testing to pick up disease risk in a person that has not presented with symptoms Reference (normal) values • • • • • The reference range is established from extensive sampling of a representative population, the results are averaged (plus or minus 2 standard deviations of the average) Range of results considered normal for a person in that population and that do not indicate disease Can be modified by age and/or gender Can be misleading! What is normal for one group is not normal for another Results must be considered in context and your doctor must know what you were doing at the time or just before the test was conducted e.g. exercising Quality control • • • Ensuring ‘control’ samples are run in parallel with test samples. Age/expiry date on samples Was your sample collected the right way for the assay? Enzyme assays • • • • Enzymes are specialised proteins that catalyse (i.e. make possible or more efficient) biochemical reactions, thereby enabling most functions we associate with life Enzyme + substrate = enzyme + product OR chain of enzymatic reactions i.e. E1+S1 = E1+P1 P1+E2 = E2+P2 We exploit the specificity of enzymes for their substrates in the laboratory Substrates or products can have a specific absorbance profile that can be measured Spectrophotometers: Colourimetric assay e.g. glucose • Pink chromogen absorbs light at 515nm Standard curve: • Temperature and concentration of reagents are important → change how efficiently the enzyme works → standard curves must be performed alongside sample analysis ELISA (enzyme link immunosorbent assay) • • Often used for the detection of protein based molecules: E.g. c-reactive protein, troponin C, apolipoprotein B, infectious agents (e.g. bacteria) and antibodies to see if you have been vaccinated (e.g. mumps) or autoimmune conditions 1. 2. 3. 4. 5. → presence of coloured product indicates that the patient has mounted an immune response to Hepatitis B Basic chromatography methods • • Separation by: o Size o Shape o Charge o Affinity to ligand or antibody The essential components of a chromatographic system are: o Stationary phase o Mobile phase o Delivery system o Detection system Stationary phase: size 1. 2. 3. Analysing the sample • As the sample is collected in ‘aliquots’ it can be analysed using standard spectrophotometer techniques i.e. different substances have different absorbance profiles for identification or quantification High performance liquid chromatography • • • • Samples are pushed through a column pack with a stationary phase under high pressure. Stationary phase is denser with smaller particles giving better resolution Different substances will take different times to get through the column A detector uses UV or visible light for qualitative or quantitative analysis of the separation Stationary phase: charge 1. 2. 3. Chromatography: affinity • Protein binds to antibody or ligand (e.g. enzyme binds to its substrate) while the nonbinding proteins are washed out Flow cytometry • • • • • Uses fluidics to create a liquid stream of single cells Uses lasers to measure the properties of individual cells Can use either fluorescent dyes or fluorescently labelled antibodies to quantify the state of a cell or the expression level of a marker Routinely used for diagnosis of haematopoietic cancers Sample collection depends on downstream analysis o Collecting blood: ▪ Temperature (keep on ice, room temp or warm) ▪ Plasma vs serum tubes ▪ Types of tubes (e.g. Heparin or EDTA coating will prevent clotting) ▪ Is centrifugation needed to separate the layers of blood – want unclotted blood ▪ Quantity