CCHM321-Lesson 4&5.pdf

CLINICAL CHEMISTRY 01 CCHM 321 |THIRD YEAR | 1ST SEMESTER | 2024 – 2025 PRELIM | WEEK 3...

CLINICAL CHEMISTRY 01 CCHM 321 |THIRD YEAR | 1ST SEMESTER | 2024 – 2025 PRELIM | WEEK 3 THRYNT MIRANDA NUCUM LESSON NO. 3 | LABORATORY AUTOMATION AND TECHNIQUES DESIGNS AND TERMINOLOGIES DESIGNS TOPIC OUTLINE 1 Sequential Analyzer 1 Laboratory Automation - One test at a time 2 Driving forces and Benefits of Automation 3 Designs and Terminologies 2 Batch Analyzer 4 Four main types of automatic analyzers - One test but for multiple specimen 5 General function of automated analyzers 6 Quantitating the Reaction Result 3 Parallel Analyzer 7 Definition of term - Numerous tests but for single specimen 4 Random Access Analyzer LABORATORY AUTOMATION - Many tests in many specimen Performs tests in any order Laboratory Automation - is characterized by automating, using robotics and instrumentation to do tasks that were traditionally manually performed by humans. TERMINOLOGIES - Many steps in the total testing process can now be performed automatically 1 Sequential Testing permitting the laboratorians to focus on manual or technical - Multiple tests analyzed one after the other on a given specimen processes that increase both efficiency and capacity. 2 Batch Testing THREE MAJOR PHASES OF TESTING PROCESS - All specimens are loaded at the same time and a single test is Pre Sample Processing/Preparation conducted on each sample Analytical Preparation of the sample for analysis has been and remains a 3 Parallel Testing manual process in most laboratories. - More than one test on a given sample Analytical Analyte Measurement 4 Random Access Testing - Any test can be performed on any sample in any sequence Actual process of measuring and testing of analytes 5 Open Reagent System Post Includes: - System other than the manufacturer’s reagent can be used Analytical a) Reporting of result Laboratory can use reagent of different supplier b) Specimen storage and retrieval c) Data management 6 Closed Reagent System - Operator can only use the manufacturer’s reagent Exclusively use the reagent of manufacturer TOTAL LABORATORY AUTOMATION Total Laboratory Automation FOUR MAIN TYPES OF AUTOMATIC ANALYZERS - The comprehensive automated systems 1 Continuous Flow System Interconnection of the three major phases of testing process 2 Discrete Sampling Analyzers 3 Centrifugal Fast Analyzers 1 Steps in the pre-analytic phase can be automated by either stand-alone 4 Thin – film Analyzers (Dry Slide Analyzers) systems or systems that connect to the analytical modules with tracks that transport the specimens. 1 CONTINUOUS FLOW SYSTEM 2 Post- analytic refrigerated storage and retrieval systems can also be PRINCIPLE integrated with pre-analytic and analytic automation. Continuous Flow System - Liquids are pumped into a system of continuous tubing DRIVING FORCES AND BENEFITS OF AUTOMATION Reagents 1 Rapid Results Diluents analyzers are faster and easier to use → as a result of Samples continuous reengineering and electronic refinements. generate results in a matter of seconds A samples are introduced in a sequential manner following each other in a single network 2 Increase in the number of tests performed machine has multiple testing capacity B all samples are carried through the same analysis pathway 3 Saves time and effort C all samples automatically pass from on step to another without Walk-away capability waiting to bring the samples to the same stage of completion 4 Elimination of needs for personnel D Reactions are not necessarily carried to equilibrium since samples Minimal operator intervention (1 MT for many tests) and standards are treated exactly alike E Parallel Single Channel ADVANTAGES OVER MANUAL PROCEDURES - Multiple of test on each sample 1 Economical small, portable, easy-to-operate benchtop analyzers single test is minimized FEATURES A Use of: Low cost 1) Plastic tubes of different diameters 2) Peristaltic pump 2 Errors in calculations and transcriptions are reduced Less human Variation - used for continuous pumping of the samples and reagents - replaces the pipetting steps aspirates the sample from the tubes 3 Better precision and accuracy Dependent on the reliability of the machine B Introduction of Air Bubbles at a regular interval 4 Increased safety separate the sample and reagent into segments 5 Reduction of specimen volume requirement separate one sample from the next 6 Partially alleviates personnel requirement continuous scrubbing of tubing → for cleaning purposes prevents cross contamination DISADVANTAGES OF AUTOMATION 1 There may be limitation in the methodology that can be used C Dialyzers Specimen collection and preparation by MT - removes the protein from the reaction - prevents interferences caused by proteins 2 MT is often discouraged from making observations and using their own judgements about potential problems D Recorded read-out - used for easy retrieval of results 3 Many systems are impractical for small number of samples Machines are best used for significant number of samples E Modular Design permitting interchanging of major parts - to easily replace of parts in case of malfunction 4 Expensive to purchase and maintain Worth millions THRYNT 1  3 CENTRIFUGAL FAST ANALYZERS PARTS 1 SAMPLER PRINCIPLE A As the rotor is accelerated, centrifugal force moves the reagents and Sampler sample to a mixing chamber and then through a small channel into the - Holds the cups containing the standards and specimens for analysis, cuvette. which are introduced into the analytical system by means of aspiration, in a preset sequence and a pre-selected rate B As the filled cuvette rotates past a fixed light beam, the absorbance of the reaction is measured spectrophotometrically. Example: Cuvette in spectrophotometer C Mass analysis is an advantage as reactions in cuvette are made virtually simultaneously 2 PUMPS AND MANIFOLDS D Uses the force generated by the centrifugation to transfer and then Pumps and Manifolds contain liquids in separate cuvette for measurement at the perimeter of - for continuous and proportional delivery of samples, reagents, or gases a spinning rotor - analogous to pipetting in manual techniques E Laboratory with high workload for individual test for routine batch machine aspirates the samples and deliver it to the system analysis may use this 3 DIALYZERS Most capable of running multiple samples, one test at a time (Batch Testing) Dialyzers - Employs dialysis through a semi-permeable membrane to separate EXAMPLES proteins from the analytes, thus eliminating the need for manual 1 CentrifiChem deproteinization techniques. 2 RotoChem eliminates the use of trichloroacetic acid for manual testing 4 THIN FILM ANALYZERS 4 HEATING BATH PRINCIPLE Heating Bath A 16 mm square chip which contains several very thin layers, accepts a - For heating and incubating the reaction mixture and fixed temperature metered drop of serum, spreads it evenly into a reagent layer, then confines (37°C) the colored product to the fixed area for reflectance spectrophotometry. Examples: EXAMPLES OF CONTINUOUS FLOW ANALYZERS Kodak “EktaChem” Technicon capable of running 3 different tests at 60-80 samples per Analyzers II hour SMA 6/60 Capable of running 6 different tests at 60 samples per hour GENERAL FUNCTIONS OF AUTOMATED ANALYZERS SMA 12/60 Capable of running 12 different tests at 60 samples per hour 1 Collection and Preparation of the sample - Use of bar-coded labels on the samples which allow identification of SMAC capable of running 40 different tests at 120 samples per the sample and the tests requested hour 2 Sample and Reagent Measurement of the Sample SMA (Simultaneously Multiple Analyzer) - Automates measurement measure, aspirate, and introduce samples into the analyzer reagents - Reagent and samples are combined in a prescribed manner to yield a specific final concentration MAJOR DRAWBACKS - Mixing of reagents and samples can be done by stirring, agitation, or 1 Significant carry-over problems by some other device 2 Wasteful use of continuously flowing reagents 3 Incubation 2 DISCRETE SAMPLING ANALYZER - Waiting period in which mixture is allowed to react - Done at a specified, constant temperature controlled by the analyzers PRINCIPLE A Each sample reaction is handled in a separate compartment and 4 Monitoring or Sensing the Reaction Result does not come into contact with another sample. - Some measurement can be done in the vessel, cell, or cuvette where B The samples and standards are handled on a batch basis and must the reaction is done by optical, thermal, or electrical means known as be brought before proceeding to the next procedure. “situ-monitoring” C All reactions must be carried out until equilibrium is reached. D Have the capability of: Running multiple test at one sample at a time (Parallel QUANTITATING THE REACTION RESULT Testing) Digital - Usually restricted to certain mathematical functions Multiple samples; One test at a time (Batch Testing) Computation (such as addition or subtraction) - The converter changes the voltage or current signal into Most popular and versatile analyzers a digital from which can then be processed by the - Have almost completely replaced the continuous flow and centrifugal computer. analyzers because each sample is in a separate reaction container, leading to quality in each cuvette and particular sample is not affected Analog - Uses an electrical signal from the sensor, as from the Computation photoelectrical cell and compares it with a reference signal as for the blank solution EXAMPLES OF DISCRETE SAMPLING ANALYZERS Dupont ACA Reagents of each test are packaged in a special pack with VISUALIZING THE RESULT a rigid header 1 visualizing the instrument read-out is with the use of television monitor This pack serves as the reaction chamber and (cathode-ray tube) or light emitting diodes test cuvette for photometric analysis 2 visualized read-out can be converted to a hard copy by means of a paper or Each test pack contains: tape print-out Chromatographic column Gel filtration matrix 3 data print-out information is transferred or transcribed to laboratory result Precipitant slips or other permanent records OTHER EXAMPLES 4 if results are interfaced with a laboratory computer, this transcription 1 ABBOTT ABA-100 BIOCHROMATIC ANALYZER, ABA-200 and VP process is done quickly and without errors, which may occur when ANALYZER transcription is done manually - Equipped with a single disposable plastic 32 compartment, water bath, uses ultra microsamples, and bichromatic light to resolve STANDARDIZATION problem with interference A frequent standardization of methods usually done before the start of the day B once standardization has been done, a well-designed automated system 2 BECKMAN ASTRA 8 and ASTRA 4 maintains or reproduces the prescribed conditions with great precision - microprocessor controlled instrument, uses ultra microsample, results are displayed on a screen, multichannel analyzer in effect 3 BECKMAN DSA and DSA 564 - offer double beam photometers for automatic blank correction, micro amount of sample 4 AMERICAN MONITOR KDA - computer controlled, single channel analyzer, results are stored with subsequent print-out of collated patient results. THRYNT 2  DEFINITION OF TERMS A Test Repertoire - Number of tests that can be performed on the instruments B Selective - Machine only performs the test required C Dwell Time - Minimum time required to obtain the result after the initial sample of the specimen D Throughput - Maximum number of samples that can be processed within an hour Measure of an analytical system E Cost - Reagent’s maintenance, calibration, quality control, and capital F Test Menu - List of all the tests performed by a particular laboratory, exclusive of those tests it sends to reference laboratory G Workload - Number of test result that are generated by a laboratory during a given time period H Walk – away capability - Ability of the operator to program the instrument to perform other tasks while the instruments are processing the test I Bar Code - Means of providing positive sample in identification J Linearity - linear range is generally defined by the values of the highest and lowest calibrations available for a particular instrument K Sensitivity - lowest value that can be reliably detected without providing a false positive result measures how often a test correctly generates a positive result for people who have the condition that’s being tested for (a.k.a. true positive rate) L Specificity - measures only the analyte requested M Shelf Life - reagent’s stability before use N Carry Over - system where we used cuvette that are insufficiently washed for each testing cycle O Maintenance Time - time that analyzers are not been used P Downtime - time that analyzers are not available for the testing because of periodic maintenance or reasons leading to troubleshooting Q Flags - warning or malfunctions R Laboratory Automation - multi-disciplinary strategy to research, develop, optimize, and capitalize on technologies in the laboratory that enables new and improved processes S Reflex Testing - use of preliminary test results to determine if additional tests should be requested or cancelled T Dead Volume - amount of serum that can’t be aspirated by analyzer THRYNT 3  CLINICAL CHEMISTRY 01 CCHM 321 |THIRD YEAR | 1ST SEMESTER | 2024 – 2025 PRELIM | WEEK 4 THRYNT MIRANDA NUCUM LESSON NO. 4 | ANALYTICAL METHOD AND INSTRUMENTATION BEER’S LAW TOPIC OUTLINE Beer’s Law 1 Electromagnetic Energy - Also called as “Beer-Lambert’s Law” 2 Wavelength - States that the concentration of a substance is directly proportional to the 3 Colorimetry amount of the light absorbed or inversely proportional to the logarithm of 4 Beer’s Law transmitted light. 5 Components of Spectrophotometer 6 Quality Control TRANSMITTANCE 7 Double Beam Spectrophotometer Transmittance (T) - Also called as “Percent Transmittance” ELECTROMAGNETIC ENERGY - is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I). Electromagnetic Energy - Radiant energy from short wavelength gamma rays to long wavelength ABSORBANCE radio waves They are photons of energy travelling in a wavelength manner Absorbance (A) The shorter the wavelength, the lighter the electromagnetic - amount of light absorbed energy - It cannot be measured directly by a spectrophotometer but rather is TYPES OF ELECTROMAGNETIC ENERGIES mathematically derived from %T as follows: 1 Cosmic Rays 2 Gamma Rays 3 X-rays 4 Ultra Violet (UV) 5 Visible 6 Infrared (IR) - According to Beer’s law, absorbance is directly proportional to 7 Radio, TC, Microwave, etc. concentration ENERGY Energy Where: - Transmitted via electromagnetic waves characterized by frequency and ε = molar absorptivity wavelength A = abc Where: WAVELENGTH A = absorbance a = molar absorptivity (absorptivity of the compound under standard Wavelength conditions) - distance between peaks as light is envisioned to travel in wavelike manner. b = length of light through the solution (light path) c = concentration of absorbing molecules/solution Kinds of Wavelength Visible Spectra 340 – 700 nm Invisible Spectra 700 (Infrared Region) 1 Light Source 2 Entrance Slit Frequency 3 Monochromator - Number of vibrations of wave motion per second 4 Exit Slit 5 Cuvette 6 Photodetectors 7 Meter 1 LIGHT SOURCE Light Source - Provides radiant energy in the form of visible or non-visible light that may pass through the monochromator. The light of proper wavelength will be made incident on the analytical cell Tungsten Light Bulb (Tungsten-iodide lamp) most common source of light for work in the visible and near- infrared regions COLORIMETRY LASER (Light Amplification by Stimulated Emission of Radiation) is based on the interaction of radiant energy with suitably excited atoms or molecules. Two Primary Consideration in every Colometry Analysis: 1 Quality of Color Factors in Choosing Light Source 2 Intensity of the Color 1) Range 2) Spectral Distribution within the range Kinds of Colorimetry 3) Source of radiant production 1 Visual Colorimetry 4) Stability of the radiant energy - Uses the eye in determining the end point 5) Temperature 2 Photoelectric Colorimetry - a technique used to determine the concentration of a substance in Types of Light Source a solution by analyzing the intensity of light transmitted through the 1 Tungsten Iodine lamp solution - produces energy wavelength from 340 to 700 nm (visible region). a) Spectrophotometry 2 Quartz Halide lamp measurement of light intensity in a much narrower - contains small amounts of halogen such as iodine to prevent the wavelength. It uses a device (prisms and/or gratings) decomposition of the vaporized tungsten from the very hot to disperse the source of light into a continuous filament. spectrum. Spectrophotometric measurements 3 Deuterium Discharge lamp - provides energy source with high output in the UV range (down to b) Filter Photometry 165 nm). measurement of light intensity of multiple wavelengths. It uses filter to isolate part of the spectrum. 4 Infrared Energy source Photometric measurements - used above 800 nm Examples: Merst glower an electrically heated rod of rare earth element oxides. Globar uses silicon carbide THRYNT 1  7 METER Mercury Vapor lamp exits narrow bands of energy at well-defined places in Meter the spectrum (UV and visible) - simplest method of displaying output of the detection system. Also called read-out device Hollow Cathode lamp Galvanometer/ammeter consists of a gas-tight chamber containing anode, a cylindrical cathode, and inert gas such as helium of argon. QUALITY CONTROL Didymium and holmium oxide filter 2 ENTRANCE SLIT - used to check wavelength accuracy Entrance Slit Neutral density filters and dichromate solution - minimizes unwanted or stray light and prevents the entrance of scattered - verify absorbance accuracy on linearity light into the monochromator system ➔ Stray light causes systematic error – causes loss of linearity DOUBLE BEAM SPECTROPHOTOMETER 3 MONOCHROMATOR Double-Beam Spectrophotometer - Splits monochromatic light into two components Monochromator - Isolation of individual wavelengths of light is an important and necessary Two Types: function of a monochromator. 1) Double-beam in space uses 2 photodetectors, for the sample beam and reference Types of Monochromator beam 1 Prisms - wedge-shaped pieces of glass, quartz, NaCl, or some other 2) Double-beam in time material that allows transmission of light. uses one photodetector and alternately passes the - Disperses white light into a continuous spectrum of colors based monochromatic light through the sample cuvette and then on variation of refractive index for different wavelength. reference cuvette using a chopper 2 Gratings - has small grooves cut at such as angle that each groove behaves like a very small prism. - Separates white light into various color component. - Based on the principle that wavelengths are bent as they pass a sharp corner. 3 Colored filters - made of a glass that absorbs some portion of the electromagnetic spectrum and transmit others. - Light energy is absorbed by dye compounds on the glass and is dissipated as heat. 4 Interference filter - utilizes the wave character of light to enhance the intensity of the desired wavelength by constructive interference and eliminates others by destructive interference and reflections. 4 EXIT SLIT Exit Slit - controls the width of light beam (bandpass; accurate→