Clinical Chemistry - 05 Instrumentation

CLINICAL CHEMISTRY WAVELENGTH INSTRUMENTATION • OUTLINE • Analytical Methods • Colorimetry o Electromagnetic Radiation/Energy o Spectrophotometry o Flame Photometry o Atomic Absorption Spectrophotometry (AAS) • Volumetric Method • Turbidimetry • Nephelometry • Electrophoresis o Components of Electrophoresis o Factors affecting Migration Rate o Densitometry o Isoelectric Focusing • Chromatography o Forms of Chromatography o Mass Spectrometry (MS) o Gas Chromatography-Mass Spectrometry (GC-MS) o Liquid Chromatography-Mass Spectrometry (LCMS) o Separation Techniques Used in Liquid Chromatography • Fluorometry o Components of Fluorometry o Factors influencing Fluorometry • Chemiluminescence • Osmometry o Principle of Osmometry o Freezing-Point Depression Osmometry • Electrochemistry Techniques o Potentiometry o Coulometry o Amperometry o Voltametry • Surface Plasmon Resonance • Point-of-Care Testing • Automation (Clin.Chem.) o Basic Approaches to Automation o Dry-slide Technology • Terminologies • • • The distance between two successive peaks (expressed in nanometer). <400 nm – Ultraviolet region (invisible region) 400-700 nm – visible spectrum >700 nm – Infrared region (invisible) ANALYTICAL METHODS • • • • • • • Colorimetry Volumetric Method Turbidimetry Nephelometry Electrophoresis Chromatography Fluorometry • • • • • • Chemiluminescence Osmometry Electrochemistry Techniques Surface Plasmon Resonance Point-Of-Care Testing Automation In Clinical Chemistry FREQUENCY COLORIMETRY • The number of vibrations of wave motion per second. ELECTROMAGNETIC RADIATION/ENERGY • • Photons of energy travelling in waves. Relationship between energy and wavelength is described by Planck’s Formula: E = hv o E – energy of photon (joules, EV), o h – constant (6.626 x 10-34 erg sec) o v – frequency ENERGY, WAVELENGTH AND FREQUENCY SPECTROPHOTOMETRY • • • • Most commonly used instrument for measurement of analytes. Measure the light intensity in a narrower wavelength. Involves measurement of the light transmitted by a solution to determine the concentration of the lightabsorbing substances in the solution. PARTS OF SPECTROPHOTOMETER • • Light Source o aka Radiant Source o Most important part o Provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest. o Continuum source ▪ Emits radiation that changes in intensity ▪ Widely used in the laboratory ▪ Examples: ▪ Tungsten-iodide lamp – most common • aka Incandescent tungsten lamp • provides visible and infrared regions (up to 1200nm) ▪ Deuterium lamp – provides UV radiations (down to 165nm) ▪ Xenon discharge lamp – provides visible and UV spectra of wavelength/radiations. o Line Source ▪ Emits limited radiation and wavelength. ▪ Examples: ▪ Mercury Arc Lamp, Sodium Vapor Lamps – UV and visible region, respectiveley ▪ Hollow Cathode Lamp – used for atomic absorption spectrophotometry ▪ LASER - various types provide different radiation. • Light Amplification by the Stimulated Emission of Radiation o Important Factors: ▪ Range ▪ Spectral distribution within range ▪ Source of radiant production ▪ Stability of radiant energy ▪ Temperature Entrance Slit o Minimized stray light o Prevents entrance of scattered light into the monochromator. o Stray Light: ▪ aka Unwanted light ▪ any wavelengths outside the band transmitted by the monochromator. ▪ light not originated from light source. ▪ most common cause of loss of linearity at highanalyte concentration. • Causes absorbance error • • • Monochromator o Isolates specific or individual wavelength of light from the light source o Kinds: ▪ Prisms, Diffraction Gratings, Filters o Prisms ▪ Wedge-shaped piece of glass, quarts or sodium chloride ▪ Shorter wavelengths are refracted more than longer wavelengths ▪ Can be rotated to allow only the desired wavelength to pass through. o Diffraction Gratings ▪ Most commonly used ▪ Made by cutting grooves (parallel grooves) or slits into an aluminized surface of a flat piece of crown glass ▪ Wavelengths are bent as they pass a sharp corner (diffraction - lines are bent). o Filters ▪ Simple, least expensive, not precise but useful ▪ Light waves enter one side of the filter and are reflected at the second surface (constructive interference of waves) ▪ Allows wide band of radiant energy and have low transmittance of the selected wavelength. Exit Slit o Controls the width of light beam (bandpass) o Only allows a narrow fraction of the spectrum to reach the sample. o Bandpass – total range of wavelength transmitted by the monochromator o Accurate absorbance – bandpass of < 1/5 the natural bandpass of the spectrophotometer. Cuvet o aka absorption cell, analytical cell, sample cell. o It holds the solution to be measured. Sample holder o Dissolved by prolonged contact with alkaline solution producing etching which could affect light absorbance o Cuvets with scratch should be discarded ▪ Scratches scatters light o Kinds: ▪ Alumina Silica Glass, Quartz/plastic, Borosilicate Glass, Soft Glass o Alumina Silica Glass ▪ Most commonly used (can be used in 3502000nm) ▪ Effectively transmit light at ≥220 nm. o Quartz/plastic ▪ Used for measuring solutions requiring visible and spectra. o Borosilicate Glass o Soft Glass Photodetector o Detects the amount of light that passes through the sample in the cuvet. o Detects and converts transmitted light into photoelectric energy. o Kinds: ▪ Photocell, phototube, photomultiplier tube (PMT), photodiode o • • Photocell ▪ aka Barrier layer cell, photovoltaic cell ▪ The simplest detector ▪ Composed of selenium or iron plate covered with transparent layer of silver • Electrons from selenium/iron plate are released to the silver layer if exposed to light ▪ Used in detecting radiation in the visible region. ▪ Maximum sensitivity: 550nm o Phototube ▪ Similar to photocell as it also gives off electron when exposed to light energy. ▪ Contains cathode & anode enclosed in glass case o Photomultiplier Tube (PMT) ▪ Most commonly used ▪ Used in detecting visible and UV regions ▪ Has excellent sensitivity and rapid response ▪ Will burn out if exposed to room light. • Must be well-covered o Photodiode ▪ With excellent linearity ▪ Most useful as a simultaneous multichannel detector. Read-Out Device o Displays output of the detection system o Examples: ▪ Galvanometer ▪ Ammeter ▪ Light-emitting diode (LED) display Review: PRINCIPLE: BEER’S LAW • • • • It states that the concentration of the unknown substance is directly proportional to the absorbed light (absorbance) and inversely proportional to the transmitted light (% transmittance). o Higher conc. of substance in an unknown, the higher light intensity it can absorb which mean lower light transmittance It mathematically establishes the relationship between concentration and absorbance. Absorbance o The amount of light absorbed o Proportional to the inverse log of transmittance o Mathematically derived from %Transmittance o Expressed as: A = abc = 2 − log%T ▪ A – absorbance ▪ a – absorptivity of compound ▪ b – length of light through the solution ▪ c – concentration of solution. Percent Transmittance (%T) o Ratio of radiant energy transmitted (T) divided by the radiant energy incident of the sample (I) o ▪ It – transmitted light thru the sample. ▪ I0 – intensity of light striking the sample. %T measured by commercial spectrophotometers: ▪ Ratio of sample transmitted beam divided by the blank transmitted beam BLANKING TECHNIQUE • TYPES OF SPECTROPHOTOMETERS • • Single-beam Spectrophotometer o Simplest type o Designed to make one measurement at a time at one specified wavelength. o The absorption maximum of the analyte must be known in advance when a single-beam instrument is used Double-beam Spectrophotometer o Type that splits monochromatic light into two components: ▪ One beam that passes through the sample ▪ Other through a reference solution/blank • To correct for variation in light source intensity. o The absorbance of the sample can be recorded directly as the electrical output of the sample beam. o Double-beam in space ▪ Has 2 photodetectors: • sample beam • reference beam o Double-beam in time ▪ Has one photodetector and alternately passes themonochromatic light through the sample cuvet and then reference cuvet using a chopper or rotating sector mirror. • • • Used when the reageant to be used in the method has color already (eg. Creatinine, albumin) The darker the color of solution, the higher the absorbance Reagent Blank – used to correct absorbance caused by color of reagents. o After reagent blank, the absorbance of reagents is automatically subtracted from the unknown reading o Colored reagent may interfere with the absorbance Sample Blank – measures absorbance of sample and reagent in the absence of the end-product. o Corrects absorbance for interferences absorbing the wavelength of measurement. (eg. Hgb) FLAME PHOTOMETRY • • • • • • • aka Flame Emission Photometry. Measures light emitted by a single atom burned in a flame. Principle: Excitation of electrons from lower to higher energy state. Components: o Light Source: Flame (also serves as the cuvet) o Monochromator: Filter o Photodetector: Photocell Method: Indirect Internal Standard Method Internal Standard: Lithium (preferred) or Cesium o It corrects variations in flame & atomizer characteristics Purpose: measurement of excited ions (Na+ & K+) ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS) • • • • • • • It measures light absorbed by atoms dissociated by heat. Principle: element is not excited by merely dissociated from its chemical bond and place in an unionized, unexcited, ground state. Purpose: measurement of unexcited ions (Ca2+ & Mg2+) o Reference method for measurement of Ca2+ & Mg2+ Interference: Phosphate - interferes w/ Ca2+ measurement ▪ Strontium/Lanthanum Chloride: added to solution to form complex with phosphate. Components: o Light source: Hollow-cathode lamp o Monochromator: Diffraction gratings, prism o Photodetector: Photomultiplier tube (PMT) More sensitive than flame photometry (due to PMT) Zeeman Effect o Background correction technique for AAS. o The presence of intense static magnetic field will cause the wavelength of the emitted radiation to split into several components (shift in wavelength) VOLUMETRIC METHOD • • • aka Titrimetric Method Principle: Unknown sample is made to react with a known solution in the presence of an indicator Examples: Schales and Schales method (chloride test), EDTA titration method (Calcium test) TURBIDIMETRY • • • • • For measuring abundant large particles (proteins) and bacterial suspensions. Principle: the amount of light blocked by a particulate matter suspended in a turbid solution. (↑ MW = ↑ blocked) Dependent on specimen concentration and particle size. Uses spectrophotometer for quantitation Uses: o Protein measurement o Detection of bacterial growth (broth cultures) o Antimicrobial test (broth method) o Detect blot formation • • Iontophoresis – migration of small charged ions Zone electrophoresis - migration of charged macromolecules COMPONENTS OF ELECTROPHORESIS • • • • Electrical power Support Medium Buffer o Serum Protein Electrophoresis (SPE): Barbital (Veronal) – pH 8.6 Sample and Detector SUPPORT MEDIUM • • • Cellulose acetate o Separates by molecular size o Most commonly used in SPE (Serum Protein Electro.) o Only separate proteins into 6 fractions Agarose gel o Separates by electrical charge o Does not bind protein o Only separate proteins into 6 fractions Polyacrylamide gel o Separation is based on charge and molecular size. o Separates proteins into 20 fractions o Used to study individual proteins (isoenzymes) STAINS • • • 1. 2. 3. 4. 5. 6. 7. Used to visualize the bands Protein & enzyme electrophoresis - Amido Black & Ponceau S Lipid electrophoresis - Oil Red O, Sudan Black, Fat Red 7B Amido black Ponceau S Oil Red O Sudan Black Fat Red 7B Coomasie Blue – CSF Protein Electrophoresis (multiple Sclerosis) Gold/Silver Stain – very sensitive (nanogram of proteins) NEPHELOMETRY • • • • • • • Measures the amount of antigen-antibody complexes. Principle: it determines the amount of scattered light by a particulate matter suspended in a turbid solution. Dependent on wavelength and particle size. Use: protein measurement (1-10 mg/dL serum protein) More sensitive than turbidimetry Photodetector: Photomultiplier tube (highly sensitive) Uses nephelometer. ELECTROPHORESIS • • • Most important analytical method used to differentiate enzymes with isoenzymes. Principle: Migration of charged particles in an electric field. It separates proteins based on their electric charge density. o Cation(+) move toward electrode w/ (-) charge (Cathode) o Anion (-) move toward electrode w/ (+) charge (Anode) FACTORS AFFECTING MIGRATION RATE 1. 2. 3. 4. 5. Net charge of molecules (↑ Net charge = Faster migration) Size and shape of molecules (↑ MW = ↓ Migration Rate) Electrical field strength Nature of the supporting medium Temperature of operation. DENSITOMETRY • • It measures the absorbance of stain o Concentration of dye is directly proportional to protein fraction It scans and quantitates electrophoretric pattern ISOELECTRIC FOCUSING • • • • • Separation of molecules by migration through pH gradient. Ideal for separating proteins of identical sizes but with different net charges Principle: Proteins move in the electric field until they reach a pH equal to their isoelectric point. o Isolectric point - pH where proteins are neutral pH gradient - created by adding acid to the anodic area and adding base to the cathode area. CHROMATOGRAPHY • • • Thin Layer Chromatography o Semiquantitative drug screening test. o Components of sample are identified by comparison with standards on the same plate. o Samples: blood, urine, and gastric fluid. o Sorbent (Stationary phase): thin plastic plates impregnated with a layer of silica gel or alumina o Based on retention factor (Rf) value. ▪ The distance a component migrates compared with the distance the solvent front moves. ▪ Each drug has a characteristic Rf value and it must match the Rf value of the drug standard. A group of techniques used to separate complex mixtures based on the physical interaction between the individual compound and the stationary phase of the system. Principle: Separation of soluble components in a solution by differences in physical-chemical characteristics of constituents. Basic Components: o Mobile phase (gas or liquid) – flows though the stationary phase; solvent o Complex mixture (sample) – dissolved in the mobile phase o Stationary phase - depend on the chromatography COLUMN CHROMATOGRAPHY • FORMS OF CHROMATOGRAPHY • PLANAR CHROMATOGRAPHY • Paper Chromatography o Used for fractionation of sugar and amino acid o Sorbent (stationary phase) – Whatmann paper Gas Chromatography o Used for separation of steroids, barbiturates, blood, alcohol, and lipids. o Useful for compounds that are volatile or easily converted into volatile form o Elution order of volatiles is based on their boiling point. o Mobile phase: nitrogen, helium, hydrogen, and argon (inert gases) o Two Types: o Gas Solid Chromatography – separation occurs based on differences in absorption at the solid phase surface o Gas Liquid Chromatography – separation occurs by differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase. Liquid Chromatography o Based on the distribution of solutes between a liquid mobile phase and a stationary phase. o High Performance Liquid Chromatography (HPLC) ▪ The most widely used liquid chromatography ▪ It uses pressure for fast separation, controlled temperature, in-line detectors and gradient elution technique ▪ Use: • fractionation of drugs, hormones, lipids, carbohydrates, and proteins • Separation and quantitation of various hemoglobins associated with specific diseases (Thalassemia). • Rapid HbA1c test (w/in 5 minutes) MASS SPECTROMETRY (MS) • • Based on the fragmentation and ionization of molecules using a suitable source of energy Can detect structural information and determination of molecular weight. • • Tandem mass spectroscopy – can detect 20 inborn error of metabolism from a single blood spot o Method of choice for newborn screening In tandem with Chromatography: GC-MS or LC-MS. o Chromatography – separation of components. o Mass Spectrometry – quantify the compounds separated by chromatography. SEPARATION TECHNIQUES USED IN LIQUID CHROMATOGRAPHY • GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) • • • • • • • The gold standard for drug testing Used for xenobiotics, anabolic steroids, pesticides In this method, quantitative measurement of drug can be performed by selective ion monitoring. The drug is split into component ions be electron beam (decomposition fragments) The position of the parent molecule ion and degradation fragments give rise to fingerprint pattern. o Position of parent molecule - area where sample is put o Degradation fragment - movement that occurred Each drug has its own fragmentation pattern (fingerprint) is compared to a computer library of known fragmentation pattern. General steps: o GC - separates the analyte of interest from a sample o MS - analyte will be fragmented to allow identification by comparing it to a known fragmentation library. • • • • Gel Chromatography o separates molecules based on differences in their size and shape o Hydrophilic Gel (Gel filtration) ▪ separation of enzymes, antibodies and proteins. ▪ Example: agarose, dextran o Hydrophobic Gel (Gel permeation) ▪ separation of triglyceride and fatty acids. ▪ Example: Sephadex Ion Exchange Chromatography o The mechanism is exchange of sample ions & mobilephase ions with charged group of stationary phase. o Separation of amino acids, proteins and nucleic acids. Partition Chromatography (Liquid liquid chromatography) o Separation is based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support o Separation of therapeutic drugs and their metabolites. Affinity Chromatography o Uses immobilized biochemical ligands as the stationary phase to separate a few solutes from other unretained solutes. o Uses the lock-and-key binding (specific attachment) o For separation of lipoproteins, carbohydrates, and HbA1c, antibodies Adsorption Chromatography (liquid-solid chromatography) o Separation is based on the differences between the adsorption and desorption of solutes at the surface of a solid particle. o The compounds are adsorbed to a solid support media. FLUOROMETRY • • • • • aka Molecular Luminescence Spectrophotometry It measures the amount of light intensity (fluorescence) present over a zero background Principle: It determines the amount of light emitted by a molecule after excitation by electromagnetic radiation. 1000x more sensitive than spectrophotometer. o Emitted radiation is measured directly. Uses: Porphyrins, magnesium, calcium, & cathecolamines. COMPONENTS OF FLUOROMETRY • • LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY (LCMS) • • • • • Used for detecting nonvolatile substance in body fluids. Utilized to confirm positive results from screening of illicit drugs Complementary method to GC-MS Also used in therapeutic drug monitoring, toxicology and studies of drug metabolites. It requires interface methods to convert nonvolatile to volatile compounds. o Electrospray (ES) & Atmospheric Pressure Chemical Ionization (APCI) • Light Source – Gas Discharge Lamp o Mercury arc o Xenon lamp Monochromators – 2 monochromators (prisms, filters, gratings) o Primary Monochromator – selects the wavelength – isolates light. o Secondary Monochromator – prevents incident light from striking the photodetector. Light Detector – photomultiplier tube (PMT) or phototube o PMT contribute to the sensitivity FACTORS INFLUENCING FLUOROMETRY • Quenching Effect o pH and temperature changes o Chemical contaminants o UV light changes • • • • • Stokes Shift o Difference between the maximum wavelength, excitation and emitted fluorescence. FREEZING-POINT DEPRESSION OSMOMETRY • CHEMILUMINESCENCE • Principle: the chemical reaction yields an electronically excited compound that emits light as it returns to its ground state, or that transfers its energy to another compound, which then produces emission. Does not require: o Light source o Monochromator Use: Immunoassay More sensitive than fluorometry o Photodetector: Photomultiplier tube • • ELECTROCHEMISTRY TECHNIQUES • • • • Emission of light is created from a chemical or electrochemical reaction, and not from absorption of electromagnetic energy. Emission of light when an electron returns from higher energy level (excited) to a lower energy state. The excitation is a result of chemical reaction with compounds such as: o Luminol, isoluminol, acridinium esters, luciferin, hydrogen peroxide, hypochlorite and oxygen. Which are catalyzed by: o Enzymes (Alkaline Phosphatase-ALP, Horseradish peroxidase-HRP, microperoxidase), metal ions or metal complexes (Cu2+, Fe3+, phthalocyanine complex), hemin. OSMOMETRY • • Measurement of osmolality of an aqueous solution (serum, plasma, urine). Colligative Properties: o Osmotic Pressure ▪ the minimum pressure that must be applied to a solution to prevent the flow of solvent through a semipermeable membrane. o Boiling Point ▪ Temperature at which the vapor pressure of the solvent reaches atmospheric pressure o Freezing Point ▪ Temperature at which first solid crystal of solvent forms in equilibrium with the solution o Vapor Pressure ▪ The pressure exerted by the vapor when the liquid is in equilibrium with the vapor Measurement of current or voltage generated by the activity of a specific ion. POTENTIOMETRY CHEMILUMINESCENCE • Most commonly used method for measuring the changes in colligative properties of a solution Based on the principle that addition of solute molecules lowers the temperature at which a solution freezes 1.0 mOsm/Kg solution has a freezing point depression of 0.00186 C ̊ when compared with pure solvent (water) Blood Plasma: o Osmolality: 285 mOsm/kg o Freezing point: -0.53 C ̊ • • • • • • The measurement of electrical potential due to the activity of free ions (unbound ions) It is also the measurement of differences in voltage (potential) at a constant current (reference electrode). Follows the Nernst equation Use: pH, pCO2, electrolytes Reference electrodes: o Saturated Calomel (external) o Silver-silver Chloride (internal) Concentration of ions can be calculated based from the measured potential difference between the two electrodes. ION-SELECTIVE ELECTRODE (ISE) • • An electrochemical transducer capable of responding to one given ion. It measure the electrolyte dissolved in the fluid phase of the sample in mmol/L of plasma water. Method used by most automated electrolyte analyzers It is very sensitive and selective for the ion it measures. o The ionic selectivity depends on the ion-selective membrane used. ISE membranes o The selectivity of ISE membranes are described by the Nicolsky-Eisenman equation. o Glass aluminum silicate – measure Sodium o Valinomycin gel – measure potassium o Organic liquid membrane ion exchangers – measure calcium and lithium o Glass electrode - measure pH o Gas and enzyme electrodes • Voltameter - measure diff. bet. potential and reference • • • PRINCIPLE OF OSMOMETRY • Based on measuring the changes in the colligative properties of solutions based on changes in particle concentration (osmotic pressure). o Addition of active osmotic particles to a solution increases its osmolality. o ↑ Osmolality affects four other colligative properties: ▪ ↑ Osmotic pressure ▪ ↑ Boiling point ▪ ↓ Freezing point ▪ ↓ Vapor pressure COULOMETRY • • • • The measurement of the amount of electricity (in coulombs) at a fixed potential An electrochemical titration in which the titrant is electrochemically generated and the endpoint is detected by amperometry. It follows the Faraday’s Law Use: Chloride test (CSF, serum, sweat) AMPEROMETRY • • • • • • • • • • • Measurement of current after which a potential is applied to an electrochemical cell. Detect very low analyte levels (sensitive) Anodic stippling voltammetry – measures heavy metals such as Lead and Iron. SURFACE PLASMON RESONANCE Enables to study binding of ligands to surface receptors (membrane receptor) Use to measure interactions between immobilized antibodies and freely circulating analytes. Use to measure low molecular weight compounds, proteins, biomarkers, hormones, nucleic acids, circulating antibodies, infectious organisms Disadvantage: o Expensive o Usually performed by non-laboratory personnel (not trained with quality control) TYPES OF POCT ANALYZER It is the measurement of the current flow produced by an oxidation-reaction. Use: pO2, glucose, chloride and peroxidase determination Polarography o Exact method for pO2 determination o The measurement of differences in current at a constant voltage. o Follows the Ilkovic reaction o Use: pO2, glucose. VOLTAMETRY • • Single-use, qualitative, or semiquantitive cartridge/strip tests o Urine and blood chemistry o Infectious disease agent, cardiac markers, hCG Single-use quantitative cartridge/strip tests with a reader device o Glucose o Blood chemistry o Coagulation o Cardiac markers, drugs, C-reactive protein (CRP), allergy, fertility tests o Chlamydia o HbA1c, urine albumin o Blood chemistry o pH, blood gases, electrolytes, metabolites Multiple-use quantitative cartridge/benchtop devices o Hemoglobin species, bilirubun o pH, blood agses, electrolytes, metabolites o Cardiac markers, drugs, CRP o Complete blood count AUTOMATION (CLIN. CHEM.) • • The replacement of human manipulative effort and facilities in the performance of given process by mechanical and instrumental devices that are regulated by feedback of information so that an apparatus is selfmonitoring or self-adjusting o Standardized methods o Random errors may be prevented Why automation? o Decreased turn around time o Improved accuracy and precision o Increased throughput o Eliminated human error (eg. pipetting) o Reduced workload (performing severak test at once) o Minimum sample and reagent requirements. BASIC APPROACHES TO AUTOMATION • • • Continuous Flow Analyzer Centrifugal Analyzer Discrete Analyzer CONTINUOUS FLOW ANALYZERS • • • POINT-OF-CARE TESTING • • • aka bedside testing, near-patient testing, extra-laboratory analyses, ancillary testing, physician’s office testing, alternative site testing Analytical patient testing done outside the laboratory facility. Advantages: o Fast delivery of results. o Small sample volume • • • • Liquids are pumped through a system of continuous tubing. Samples flow through a common reaction vessel. Air bubbles at regular intervals serve as separating and cleaning media. Heating bath: maintains temperature to allow complete color development (end-product) Mixing of sample and reagents: use of glass coil inserted into the flow path Disadvantage: parallel testing Example: o Simultaneous Multiple Analyzer (SMA) o AutoAnalyzer (Technicon Corp.) - 1st fully automated • Example: o Vitros, Dimension Dade, Beckman ASTRA system, Hitachi, Bayer Advia, Roche Cobas Integra, Analytics P Module, Architect II • Dry-Slide Technology o Use reflectance photometry ▪ Measurement of light reflected from a solid surface. o Do not use liquid reagent o Measures analytes by measuring the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support. o Example: Vitros CENTRIFUGAL ANALYZER • • • • • • It uses the force generated by centrifugation to transfer specimen and reagents Liquids are placed in separate cuvets for measurement at the perimeter of a spinning rotor (1000 rpm) It uses acceleration and deceleration of the rotor to transfer the reagents and sample from one chamber to another. Mixing: Centrifugal force (rotor), bubbling of air. Advantage: Batch analysis Examples: Cobas-Bio (Roche), IL Monarch TERMINOLOGIES • • • • • • • DISCRETE ANALYZER • • • • • The most popular and versatile analyzer Employs a variety of syringe pipettes to aspirate and dispense samples and reagents. Each sample-reagent mixture handled separately in its own reaction vessel. Mixing: magnetic driven teflon stirring bar, forceful dispensing, magnetic stirring bar, rotating paddle, ultrasonic energy. Advantages: o Requires 2-6 uL of sample (minimal amount) o Random access capability. o Sequential Testing Batch Testing – all samples are loaded at the same time, and a single test is conducted on each sample Parallel Testing – more than one test is analyzed concurrently on a given clinical specimen; per patient Random Access Testing – any test can be performed on any sample in any sequence (allows STAT testing) Sequential Testing – multiple tests analyzes one after another on a given specimen Open Reagent System – a system other than manufacturer’s reagent can be utilized for measurement Closed Reagent System – a system where the operator can only use the manufacturer’s reagents Pneumatic Tube Delivery System – it provides point-topoint delivery of specimens to the laboratory and offered several advantages over specimen transport by human. o Limited presence in the Philippines (St. Luke’s, High Precision)