Labortechniken: Prüfung Laborbasics Lernunterlagen PDF

# Labortechniken: Prüfung Laborbasics Lernunterlagen ## Analyseprozesse-Pekar **What is rational laboratory diagnostics?** - Optimized effort, taking into account the resulting costs, to reach the correct diagnosis. - **Stepwise diagnostics:** targeted application - Exception: Emergencies - **A...

# Labortechniken: Prüfung Laborbasics Lernunterlagen ## Analyseprozesse-Pekar **What is rational laboratory diagnostics?** - Optimized effort, taking into account the resulting costs, to reach the correct diagnosis. - **Stepwise diagnostics:** targeted application - Exception: Emergencies - **Advantages:** cost savings - **Disadvantage:** delay in diagnosis **Name the three phases of the analysis process and their characteristics!** - **Preanalytics:** Organization, patient information, sample containers and identification, sample collection, sample transport, sample preparation, preparation of reagents and analyzers - **Analytics:** Performing the actual analysis - **Postanalytics:** Report generation, report evaluation (validation), report forwarding and documentation **What is the difference between test material and sample/test material?** - **Test material:** originally obtained biological material - most important: venous blood (without 02) - **Test material:** specially prepared test material **Describe the difference between serum, plasma and whole blood!** | | Serum | Plasma | Whole blood | |---|---|---|---| | | Only plasma | Only plasma | Sum of plasma and blood cells | | Blood fluid after clotting | | Physiological blood fluid | | | | No anticoagulants | Anticoagulants | Never freeze | | | | | | | | Let stand 15-30min at room temperature | Can be centrifuged immediately | | | | Centrifuge & decant for 10 min between 30min-1h | | | Both plasma and serum are always the supernatant in the tube! **Which anticoagulants are there?** - EDTA, citrate, heparin, fluoride ## What must be considered when identifying samples? - Labeling always before collection - Barcode for additional information - Must match the requirements (written or electronic) - Labeling: name, first name, date of birth, date of collection, .... ## What must be considered when transporting samples? - Test material, no qualitative or quantitative changes - Potentially infectious - Stable and break-proof containers - Avoid postal delivery, if necessary: light-proof, break-proof, tear-proof and water-resistant, special guidelines - Cooling or heating - Packaging components: primary container, secondary container, absorbent material, outer packaging ## What are the storage options for samples? - Short-term storage: serum & plasma: 3 days in the refrigerator at 4°C - Long-term storage: freeze quickly, thaw slowly, mix well, freezer, dry ice, liquid nitrogen ## What factors can affect the analysis? - **Endogenous (unchangeable):** sex, origin - **Exogenous (changeable):** lifestyle habits, stress, diet, medication, body weight ## Name the sources of error in sample identification! - Hemolysis: red blood cells destroyed, intracellular constituents in serum/plasma - Incorrect / insufficient labeling - Sample clotting - Serum sample without waiting time - Mixing ratios and sample volume ## Describe the different analysis methods! | | Qualitative | Quantitative | Semiquantitative | |---|---|---|---| | | Is it there? | How much? | | | | positive/negative detectable/not detectable | Exact concentration with unit | Approximate concentration | | | pathological/not pathological | | Titer determination, screening tests | | | 2 decision alternatives with cut-off value (decision threshold) | | | ## Which criteria are there for evaluating an analysis method? 1. **Practicality and costs:** time requirement, safety, costs 2. **Accuracy:** best possible agreement between measured result and true value 3. **Precision:** target value verification by means of accuracy control; agreement of repeated measurements (deviations, reproducibility) -> precision control 4. **Comparability:** comparability of two methods (measuring devices, day and night shift) 5. **Analytical specificity:** ONLY the desired component is detected, false positive result = poor specificity 6. **Analytical sensitivity:** detection capability of a method, weak signals and low concentrations reliably detected = high sensitivity ## Which quality assurance options are available in postanalytics or in analytics? ### 1. Quality Assurance: - **Statistical aids:** - **Mean value** (sum of individual results : number of results) - **Standard deviation** (spread of values) - **Coefficient of variation** (standard deviation in %) - **Normal distribution** (uniform deviation from the mean value) - **Median** (lies in the middle of a sorted value set) - **Control charts** (after completion of a control cycle, new methods and quality controls, correct labeling), verification of the analysis procedure - **Precision control**, accuracy control, ring trials (ÖQUASTA) ### 2. Quality Management: - **Complete analysis process** - **In every department** - **No universally valid guidelines** - **Compliance with minimum standards** - **Participation in ring trials** (ÖQUASTA = Austrian Society for Quality Assurance and Standardization of Medical-Diagnostic Examinations) - **German guidelines:** Rili-BÄK ### 3. Classification of possible errors: - **Gross errors:** AVOIDABLE, mix-up of patient samples, incorrect pipettes, spoiled reagents, device defects - **Systematic errors:** constantly repeated errors, measure: (un)accuracy, removal of all analysis values from the true value, avoidance: accuracy controls with routine samples - **Random errors:** UNAVOIDABLE, uncontrolled fluctuations (within the measuring system and operation), measure: (un)precision, with multiple determinations pronounced spread, reduction through analytical accuracy ## How does the evaluation of analysis results work? 1. **Reference ranges:** - Basis for evaluating measurement results - Dependent on the measuring method - Guidance - Normal values: smooth transition -> no evidence of disease 2. **Validation:** - Declaration of validity of measurement results, report release - Analytical and technical validation (BMA, performance of the analysis, data of quality assurance, part of analytical evaluation before the report leaves the laboratory) - Medical Validation (LABORATORIAN, sometimes BMA, taking into account medical, patient-related factors), includes: - Transversal assessment (comparing reference ranges); - Longitudinal assessment (previous findings); - Extreme value control (extremely high/low results); - Constellation assessment (linking of findings) - Optimal validity: all patients identified (high sensitivity), all healthy persons undetected (high specificity) 3. **Report generation / Result communication:** - Content: Patient identification, test material, results, reference range and deviation - Occurs after: technical and medical validation - Form: report, partial or final report, order and sample management,... ## Tasks of the laboratory information system: - **Networked:** Work area, analysis devices - **Manages:** master data, movement data, inventory data ## Separation Techniques - Pauschenwein **Why are separation techniques needed?** - **Preparation of test materials for analysis** - Test materials rarely immediately usable, e.g. serum and plasma **Explain the different analytical separation procedures!** | Technique | Separation Reason | Result | Function | Application | |---|---|---|---|---| | Precipitation | Chemical properties (e.g. pH value) | Insoluble precipitates | Phase change between liquid and solid | Acidification of a protein solution | | Extraction | Different solubilities | Extract or extract | Soluble compounds are extracted | Coffee extraction | | Filtration | Different particle sizes | Filtrate | Filtration | Histology and cytology (tissue and cells are made visible, dyes are filtered) | | Distillation | Boiling point/vapor pressure | Distillate | separation of substances with different boiling points, one (lower boiling point) becomes gaseous and then liquefied | Distilled water | | **Sedimentation/Centrifugation** (*separation of solid constituents from the liquid to be examined*) | density | Sediment, supernatant, particle layering or fractionation | Gravity and centrifugal forces act on the tube, as strong as necessary and as gentle as possible (otherwise hemolysis), decantation complete separation after centrifugation | Blood separation, centrifugation: acceleration of the normal sedimentation process | | Electrophoresis | Particle charge, size, shape, different migration speed and direction (depending on test conditions) | | Separation in an electric field, first separation and then the same procedure is used to measure | Buffers -> separation of different proteins | | Chromatography | Different molecular properties, different distribution of individual components | | Between the stationary and mobile phase (containing the sample to be separated), first separation and then the same procedure is used to measure | Gas chromatography, liquid chromatography | ## Describe centrifugation in more detail! The mixture is rotated around its own axis-> centrifugal force, particles with high density fly further away from the axis than particles with low density. Doubling the centrifugal acceleration halves the centrifugation time! 1. **Relative centrifugal acceleration (RCA):** Each object moving in a circle at constant rotational speed is subject to an outward force! 2. **Relative centrifugal force (RCF):** - Depends on: - Rotor radius r (distance from the center of the rotor axis to the top of the vessel) - Rotational speed per minute - **Determination:** - Performance of centrifuges through calculable and achievable acceleration - **Formula:** RCF (ing) = 1,118 x 10^-5 x r x U/min^2 (RCF=Multiple of earth's acceleration; g=earth's acceleration->9,81m/s^2) - Read off using nomograms: graphical representation of the conversion ## Which centrifuge types are there? - **Swing-out centrifuge with swing-out rotors:** Metal cups are pivotable on the rotor and take a central position during rotation. - **Fixed-angle centrifuge with fixed-angle rotors:** - Rotor is a truncated cone - Only tubes without centrifugation aid - Small tubes - Fixed angle (approx. 30°) ## Name the components of a centrifuge! - Rotor/rotor head - Multiple carriers + hangers (holders for tubes) - Centrifuge chamber (closed) - Motor - Operating panel - Sometimes: cooling, vacuum system ## Which precautions should be taken? - Lid lock - Unbalance protection - Immediately remove contaminated glass and disinfect the centrifuge - Correct setting: rpm or g, start-up and run-down speed - Suitable inserts - Labeling ## Which special centrifugations are there? - **Routine:** 2000-4000 revolutions per minute - **High-speed refrigerated centrifugation:** - Up to 250,000 rpm - Cooling and high vacuum - Isolation of microorganisms, cells, cell fragments - **Ultracentrifugation:** - Up to 500,000 rpm - Cooling and high vacuum - High material requirements - **Differential centrifugation:** centrifugation in layers - **Density gradient centrifugation:** A density gradient is built up in the solvent, resulting in a layering of substances of different densities ## Measurement techniques - Pauschenwein ## Which methods of spectrometry are there? **Spectroscopy = interaction between light and matter** - **Molecular spectrometry:** absorption spectrometry/photometry (clear liquids), reflectometry, fluorometry, nephelometry, turbidimetry - **Atomic spectrometry:** atomic emission spectrometry (flame photometry), atomic absorption spectrometry - **Mass spectrometry:** Mass and charge of a particle are compared ## Explain the principle and basics of photometry! - **Measurement of electromagnetic radiation (light)** - **Measurable wavelength:** visible light (400-760nm) to UV light (<400nm) - **Polychromatic (white) light / monochromatic (monochromatic) light** - **Molecule:** electrons are excited by light -> measurement can take place wavelength/frequency=distance between two equally oscillating points - **The shorter the wavelength, the more energy-rich the radiation** - **Propagation of light:** electric component: perpendicular; magnetic component: horizontal ## Explain the principle of extinction! T S A **T = Transmission:** is transmitted; this light is measured in absorption photometry **S = Scattering:** is reflected/scattered; together with absorption, it is responsible for extinction **A = Absorption:** molecules absorb parts of the light and electrons of these molecules are excited (higher energy level); together with scattering, they are responsible for extinction **Generally:** - White light strikes glass: Molecules cause the liquid to appear green - Each chemical substance absorbs light at a specific wavelength: all wavelengths except those we see (in this case green) - The higher the optical density (absorbance) of a medium, the more opaque it is. - Only the emitted light and the received light can be measured. - The complementary color is maximally absorbed. (Use of monochromatic light) - **Absorption spectrum = wavelength spectrum** | | | | |---|---|---| | Color and wavelength of light | Complementary color | | | Red | 750-610 nm | Blueish-green | | Orange | 610-595 nm | Greenish-blue | | Yellow | 595-580 nm | Blue | | Yellow-green | 580-560 nm | Violet | | Green | 560-500 nm | Purple | | Blueish-green | 500-490 nm | Red | | Greenish-blue | 490-480 nm | Orange | | Blue | 480-435 nm | Yellow | | Violet | 435-400 nm | Yellow-green | ## How can transmission be calculated? I = emitted light T = I/Io I T = Io I C Io = incident light T = transmitted light Result always between 0 and 1 Result x 100: is given as the transmission percentage ## How is extinction (absorbance) calculated? E = -log(T) - Can be between 0 and 2 - Dimensional quantity without unit - Is given with 3 decimal places **Generally:** - Transmission: decreases exponentially with increasing density - Extinction: increases linearly with increasing density ## What does the Lambert-Beer law state? Applies only to dilute solutions and monochromatic light E = log T/ex cxd E = Extinction d = Layer thickness c = Concentration = specific molar extinction coefficient ## Describe the structure of the photometer and the individual components! 1. **Light source:** - **Continuum emitter** (continuous emission): tungsten lamp (no UV), halogen lamp (common), deuterium lamp (good UV) - **Line emitter** (only single wavelengths, not simultaneously): mercury lamp, laser (not usable), LED, xenon (immediately usable) 2. **Apertures** 3. **Monochromator:** interference filters, prism monochromators, diffraction gratings monochromators 4. **Exit slit** 5. **Cuvette:** 1 cm layer thickness, quartz glass or plastic, filling volume: macro (from 2 ml), semi-micro (0.5-2 ml), micro (up to 0.5 ml), cuvette types (see figure) 6. **Amplifier** 7. **Readout** ## Explain the principle of reflectometry! - **Principle:** for a given wavelength, the reflectivity of a test surface depends on the dye concentration present on it. **Test strip diagnostics (semi-quantitative determination):** - Use of multi-layer reagent carriers, investigation of serum, urine, whole blood - Detection is based on a color reaction - Reading of the color intensity after a defined reaction time - Light of a specific wavelength is directed onto the test field - Measurement of the reflected portion - Evaluation visually (color scale) or using a reflectometer ## Describe fluorometry / luminescence spectrometry! **Principle:** - Uses the property of some molecules to absorb light of a specific wavelength and then re-emit it at a slightly longer wavelength (lower energy). - The emitted light is measured at right angles to the incident light. **Primary radiation:** light that excites fluorescence **Secondary radiation:** light emitted by fluorescence **Chemiluminescence:** generated by substances that emit light during chemical oxidation **Bioluminescence:** generated by luminescent systems which are preceded by an energy-releasing enzymatic reaction (ATP formation) ## What are nephelometry and turbidimetry? - **Nephelometry (= scattering measurement):** The scattered light that occurs when a light beam strikes the particles of a turbid solution is measured. - **Turbidimetry (= turbidity measurement):** The attenuation of the transmitted light caused by scattering by a turbid solution is measured. ## How is density and temperature of liquids measured? - **Hydrometer and thermometer**