QC and Spectrometer .pdf

QUALITY CONTROL QUALITY CONTROL represents the techniques and procedures that monitor per performance characteristics a system of techniques to ensure with a specified degree of confidence that the result obtain from each series at analysis is true and correct QUALITY CONTROL main pur...

QUALITY CONTROL QUALITY CONTROL represents the techniques and procedures that monitor per performance characteristics a system of techniques to ensure with a specified degree of confidence that the result obtain from each series at analysis is true and correct QUALITY CONTROL main purpose is to detect and repair performance – remove sporadic spikes – to the prior chronic level, either by prompt action to restore the status quo or, preferably, by preventing the damage from occurring in the first place deals with the causes, sources of errors, mistakes and the procedures used to recognized and minimize errors; part of QA PURPOSES/OBJECTIVES OF QC 1. to check for the quality of reagents 2. to check for the stability of the machine 3. to check for technical (operator) errors INDICATIONS OF QC 1. Accuracy Refers to the degree that measurement deviates from the true or absolute value Closeness to the true value (+1 or -1) Refers to the ability of the method to determine the exact value of the substance INDICATIONS OF QC 2. Specificity Refers to the ability of the method to detect a particular substance without interference of some other substance present in the sample (eg. Benedict’s Test) 3. Sensitivity Ability of the method to detect and measure even the smallest amount of that particular substance tested for (eg. Pregnancy Test) INDICATIONS OF QC 4. Reliability Ability of the method to maintain accuracy and precision for an extended period of time or under different variables INDICATIONS OF QC 5. Precision or Reproducibility Ability of the method to give the same result on the same sample which is expressed in SD Refers to the reproducibility or closeness of values to each other May be achieved through an appropriate procedure, careful and/or trained properly INDICATIONS OF QC 6. Practicability Ability of the method to give the same result on the same sample which is expressed in SD INDICATIONS OF QC 7. Diagnostic sensitivity Ability of the analytical method to detect the proportion of individuals with the disease Indicates the ability of the test to generate more true – positive results and few false – negative Sensitivity (%) = 100 x the number of diseased individuals with a positive test total number of diseased individuals tested INDICATIONS OF QC 8. Diagnostic specificity The ability of the analytical method to detect the proportion of individuals without the disease Reflects the ability of the method to detect true – negatives with very few false – positive Confirmatory tests require high specificity to be certain of the diagnosis Specificity (%) = 100 x the # of individuals w/out the dse with a negative test Total # of individuals tested w/ the dse KINDS OF QC 1. Intralab QC (Internal QC) Involves the analysis of control samples together with the patient specimen Detects changes in performance between the present operation and the stable operation Important for the daily monitoring of accuracy and precision of analytical methods Detects both random and systematic errors in a daily basis Allows identification of analytic errors within a one – week cycle KINDS OF QC 2. Interlab QC (External QC) Involves proficiency testing programs that periodically provide samples of unknown concentrations to participating clinical laboratories Important in maintaining long – term accuracy of the analytical methods Also used to determine state-of-the-art interlaboratory performance The College of American Pathologists (CAP) proficiency program is the gold standard for clinical laboratory external QC testing CONTROL SOLUTIONS (QC MATERIALS) Accuracy of any assay depends on the control solutions, how they are originally constituted and how they remain stable overtime General chemistry assays used 2 levels of control solutions, while immunoassays used 3 levels To establish statistical QC on a new instrument or on new lot numbers of control materials, the different levels of control materials must be analyzed for 20 consecutive days For highly précised assays (with CV less than 1%) such as blood gases, analysis for 5 days is adequate. CHARACTERISTICS OF AN IDEAL QC MATERIAL: Resembles human sample Inexpensive and stable for long periods No communicable diseases No matrix effects/known matrix effects With known analyte concentrations (assayed control) Convenient packaging for easy dispensing and storage TYPES OF ERROR RANDOM ERROR Present in all measurements; due to chance Type of error that varies from sample to sample unknown sources which may increase the variability of the standard deviation and test results indeterminate error; show imprecision confirmed by computing standard deviation (SD) and coefficient of variation (CV) RANDOM ERROR Sources of error: Instrument operator and environmental conditions (variations in techniques); eg. pipetting errors mislabeling of sample temperature fluctuation improper mixing of sample and reagent dirty optics SYSTEMATIC ERROR Error that influences observations consistently in one direction (constant differences) It is a measure of the agreement between the measured quantity and the true value; shows inaccuracy; predictable, determinate error Confirmed by measure of mean Detected as either positive or negative bias SYSTEMATIC ERROR Calibration problems Deterioration of reagents and control materials Improperly made standard solutions Contaminated solutions Unstable and inadequate reagent blanks Leaky ion selective electrode (ISE) Failing instrument Poorly written procedures A. CONSTANT ERROR Refers to a difference between the target value and the assayed value Independent of sample concentration Exists when there is a continual difference between the comparative method and the test method regardless of the concentration B. PROPORTIONAL/SLOPE/PERCENT ERROR Results in a greater deviation from the target value due to higher sample concentration Exists when the difference between the test method and the comparative method values is proportional to the analyte concentration CLERICAL/TECHNICAL ERROR due to miscalculations observed in linear regression highest frequency of clerical errors occurs with the use of handwritten labels and request forms INTERPRETING QC RESULTS 1. SHEWHART LEVEY–JENNINGS CHART In control: within confidence limit; not committed an error; 1 value outside the confidence level Not in control: × Outlier × Shift × Trend OUTLIER control values outside the confidence level; highly deviating caused by random error or systematic SHIFT 6 or more consecutive values which are contributed either on one or other side of the mean Main cause: improper calibration of instrument (systematic error) TREND 6 or more consecutive values which crosses the mean in an increasing or decreasing pattern Main cause: deterioration of reagents (systematic error) 2. WESTGARD MULTIRULE INTERPRETATION 12s Violation: 1 value exceed +/- 2SD; “warning” rule; random error 13s Violation: 1 value exceed +/- 3SD; random error 22s Violation: 2 consecutive values exceed +/- 2SD; systematic error 41s Violation: 4 consecutive values above or below 1SD; systematic error 2. WESTGARD MULTIRULE INTERPRETATION 10x Violation: 10 consecutive results are on the same side of the target mean; systematic error R4S Violation: One control exceeding the +2s and another exceeding the -2s; difference or range between the highest and lowest control result within an analytical run exceed 4SD; random error NOTE: The acceptable reference limit is set at +/- 2SD. An analytical method is considered in control when there is symmetrical distribution of control values about the mean and there are few control values outside 2s control limits. If the analytical test results are not within the +/- 2SD confidence limit, run a new set of controls and repeat specimen testing. A control value between 2s and 3s is a sign of a potential problem. A control value outside the 3s would require corrective action. QUALITY ASSURANCE broad spectrum of plans, policies, and procedures that together provide an administrative structure for a laboratory’s effort to achieve quality goals In laboratory, it involves the monitoring of specimen acquisition, turnaround times, or proficiency testing of materials to determine analytic performance. ESSENTIAL ELEMENTS OF A QA PROGRAM: 1. commitment 2. facilities and resources 3. technical competence PRE – ANALYTICAL Processing the specimen Separating and aliquoting Improper patient preparation plasma; centrifuging Incorrect anticoagulant to Transporting the specimen to blood ratio the laboratory Incorrect specimen preservation Test ordering Incorrectly or missed Entering patient information interpreted laboratory requests Specimen collection; incorrect proper of draw ANALYTICAL PHASE Specimen analysis manual procedure automated instrumentation system Commercial controls In – house controls Record – keeping POST ANALYTICAL Reporting out specimen results (wrong transcription of the patient’s data and laboratory results) manual entries computer system Reference ranges Incomplete, unavailable or delayed laboratory results Physician contact DELTA CHECK difference between a patient's present laboratory result and the previous result which exceeds a predefined limit is referred to as a delta check Delta checks are investigated by the lab internally to rule out: 1) mislabeling 2) clerical error; or 3) possible analytical error SPECTROPHOTOMETRY SPECTROPHOTOMETRY Measurement of the intensity of light as selected wavelength Photometric measurement: defined originally as the process used to measure light intensity independent of wavelength Wavelength: distance that a periodic wave propagates in one period or the distance between wave crests; measured in nanometers (nm) The shorter the wavelength, the greater the energy contained in the light, and the greater the number of the photons SPECTROPHOTOMETRY Light is classified according to its wavelength: a)Ultraviolet (UV): < 400nm b)Visible light (VL): 400 – 700nm; produced a white light when combined altogether c) Infrared (IR): > 700nm Wavelength (nm) Region Name Color Observed chopper > aspirator > prism/monochromator Hollow cathode lamp: usual light source in AAS; consists of an evacuated gas tight chamber containing an anode, a cylindrical cathode, and an inert gas (eg. helium / argon) SPECTROPHOTOMETER Used to measure the light transmitted by a solution to determine the concentration of the light– absorbing substance in the solution  PARTS OF A SPECTROPHOTOMETER wavelength meter scale sample wavelength holder control light control zero control SINGLE BEAM SPECTROPHOTOMETER simplest type designed to make one measurement at a time at one specified wavelength the absorption maximum of the analyte must be known in advance when a single beam instrument is used DOUBLE BEAM SPECTROPHOTOMETER Splits monochromatic light into two components – one beam passes through the sample, and the other through a reference solution or blank The additional beam corrects for variation in light source intensity Absorbance of the sample can be recorded directly as the electrical output of the beam DOUBLE BEAM SPECTROPHOTOMETER 2 Types: a. Double – beam in space uses 2 photodetectors (sample and reference beam) b. Double – beam in time uses 1 photodetector and alternately passes the monochromatic light through the sample cuvette using a chopper or rotating sector mirror Figure 1. Components of a single beam spectrophotometer. A. Exciter lamp; B. Entrance slit; C. Monochromator; D. Exit slit; E. Cuvette; F. Photodetector; G. Light-emitting diode (LED) display) RADIANT ENERGY / LIGHT SOURCE provides the energy that the sample will modify or attenuate by absorption light is polychromatic and must generate sufficient radiant energy or power to measure the analyte of interest 2 TYPES OF RADIANT ENERGY: 1. Continuum emits radiation that changes the intensity very slowly as a function of wavelength examples: tungsten, deuterium, xenon 2. Line emits a limited number of discrete lines or bands of radiation, each of which spans a limited range of wavelengths examples: mercury, sodium vapor EXAMPLES OF LIGHT SOURCE 1. incandescent lamp, tungsten lamp, tungsten-iodide lamp most common source of light for work in the visible and near – infrared region wavelength: 360 – 950nm EXAMPLES OF LIGHT SOURCE 2. hydrogen & deuterium – discharge lamp most commonly used for UV work wavelength: 220 – 360nm EXAMPLES OF LIGHT SOURCE 3. Mercury – arc lamp wavelength: 313 – 546nm 3 types: a. Low – pressure mercury lamps: emit sharp – line spectrum; with both UV and visible light b. High pressure emit continuum from UV to the c. Medium pressure mid visible region EXAMPLES OF LIGHT SOURCE 4. Xenon lamp Provides a continuum of relatively high-intensity radiant energy over the spectral region of 250 – 800 nm Widely used for certain fluorescence applications because of its high energy output, stability of lamp flashes and higher UV and spectral output EXAMPLES OF LIGHT SOURCE 5. LASER Light amplification by stimulated emission of radiation Very useful in analytic instrumentation because of its high intensity and narrow bandwidth, and the coherent nature of its outputs Usually used in high resolution spectroscopy, kinetic studies of processes with lifetimes in the range of 10-9 to 10-12 IMPORTANT FACTORS TO REMEMBER THAT MAY ALTER THE RESULTS Range Spectral distance within the range Source of radiant production Stability of radiant energy Temperature ENTRANCE SLIT focuses light to the prism/monochromator/wavelength selector MONOCHROMATOR isolates individual wavelength of light or a portion of the spectrum emitted by the source and focuses it on the sample TYPES OF MONOCHROMATORS / FILTERS 1. colored-glass filters pass relatively wide band of radiant energy have low transmittance of the selected wavelength not precise, simple, inexpensive TYPES OF MONOCHROMATORS / FILTERS 2. interference filters produce monochromatic light based in the principle of constructive interference of waves transmit multiples of the desired wavelengths require accessory films to eliminate harmonic wavelengths can be constructed to pass a very narrow range of wavelengths with good efficiency TYPES OF MONOCHROMATORS / FILTERS 3. prisms narrow beam of light focused on a prism is refracted as it enters the more dense glass short wavelengths are refracted more than the long wavelengths, resulting in dispersion of white light into a continuous spectrum separates white light into a continuous spectrum through refraction with shorter wavelengths that are bent, or refracted TYPES OF MONOCHROMATORS / FILTERS 4. diffraction gratings most commonly used consists of many parallel grooves (15,000 or 30,000 per inch) etched onto a polished surface Diffraction: separation of light into component wavelengths, based on the principle that wavelengths bend as they pass a sharp corner produced linear spectra (called orders) in both directions from the entrance slit TYPES OF MONOCHROMATORS / FILTERS 5. holographic gratings type of diffraction grating formed by an interference-fringe field of two laser beams whose standing-wave pattern is exposed to a polished substrate coated with photoresist THE QUALITY OF THESE SELECTORS IS DESCRIBED BY THE FOLLOWING: nominal wavelength – represents the wavelength in nm at peak of transmittance spectral bandwidth – range of wavelengths above one-half peak transmittance; a.k.a. half power point / full width at half peak maximum (FWHM) bandpass – defines the range of wavelengths transmitted and is calculated as width at more than half the maximum transmittance EXIT SLIT determines the wavelength of light that will be selected by the dispersed spectrum SAMPLE / CUVETTE holds the solution (reagent + sample) to be measured must be made of material that is transparent to radiation in the spectral region of interest can be in round or square in shape: a. square: plane–parallel optical surfaces and a constant light path; less error from the lens effect, orientation in the spectrophotometer and refraction b. round: difficult to manufacture; sometimes not in uniform diameter KINDS OF CUVETTE: 1. Alumina silica – most commonly used (350 – 2000nm) 2. Quartz/plastic – used for measurement of solution requiring visible and UV spectra 3. Borosilicate glass SAMPLE CELL / CUVETTE Inexpensive glass cuvettes: used in visible range but they absorb light in the UV region Note: Cuvettes with scratched optical surfaces scatter light and should be discarded. PHOTODETECTORS Convert the transmitted radiant energy into an equivalent amount of electrical energy (energy to electricity) the more light transmitted, the more energy, and the greater the electrical signal that is measured TYPES OF PHOTODETECTORS 1. Visual Observation 2. Barrier – layer cell / photocell / photovoltaic cell Composed of a film of light–sensitive, semiconductor material (selenium) on a plate of flat cooper or iron Used for detecting and measuring radiation in the visible region Maximum sensitivity: 550 nm Advantages: Least expensive, rugged, require no external source of electrical energy Disadvantages: low sensitivity and fatigue TYPES OF PHOTODETECTORS 3. Vacuum phototube Has a semicylindrical cathode (negatively charged; eg. rubidium / lithium) which acts as resistor in dark but emit electrons when expose to light and a wire anode (positively charged) sealed inside an evacuated transparent envelope The concave surface of the electrode support a layer of photoemissive material that tends to emit electrons when it is irradiated TYPES OF PHOTODETECTORS 3. Vacuum phototube Difference from photocell: outside voltage is required for operation Photocurrent is linear with the intensity of the light Vacuum within the tubes avoids scattering of the photoelectrons by collision with gas molecules TYPES OF PHOTODETECTORS 4. Photomultiplier tube (PMT) Detects and amplifies radiant energy; accomplished by using multiple dynodes (series of anodes) positioned throughout the PMT Most common type pf photodetector; commonly used when radiant power is very low, which is a characteristic of very low analyte concentrations TYPES OF PHOTODETECTORS 4. Photomultiplier tube (PMT) Highly sensitive to UV and visible radiation (200 times more sensitive than the phototube); very fast response times Limited to measuring low power radiation because intense light causes irreversible damage to the photoelectric surface TYPES OF PHOTODETECTORS 5. Photodiode Absorption of radiant energy by a reverse–biased pn–junction diode. Produces a photocurrent that is proportional to the incident radiant power Advantages: excellent linearity (6-7 decades of radiant power), speed, small size TYPES OF PHOTODETECTORS 5. Photodiode Disadvantage: Not sensitive as PMT due to its lack of internal amplification Silicon Diode Transducers: more sensitive than vacuum phototubes but less sensitive that PMTs; spectral ranges from 190 – 1100 nm TYPES OF PHOTODETECTORS 6. Multichannel Photo Transducers Consist of an array of small photoelectric sensitive elements arranged linearly or in a 2D pattern on single semiconductor chip TYPES OF MULTICHANNEL PHOTO TRANSDUCERS a. Photodiode arrays (PDAs) produce a linear (one-dimensional) array of several hundred photodiodes set side-by-side on a single integrated chip (IC) or chip lower dynamic range and high noise; most useful as a simultaneous multichannel detector (Skoog, 1998) PDA detection occurs in 3 stages: (1) initialization (2) accumulation of charge at each pixel–integration time (3) read–out signals TYPES OF MULTICHANNEL PHOTO TRANSDUCERS b. Charge–injection devices (CIDs) c. Charge–coupled devices (CCDs) multichannel devices having dynamic range and a signal–to–noise ratio that are superior to the noise of PMT SIGNAL PROCESSORS / READOUT DEVICE / METER Displays output of the detection system Examples: digital meters light emitting diodes (LEDs) Galvanometers cathode–ray tubes (CRTs) d’ Arsonval meters liquid crystal displays Recorders (LCDs) BEER’S LAW The concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. Absorbance: amount of light absorbed; proportional to the inverse log of transmittance; mathematically derived from %T 1. A = 2 – log % T 2. A = ε x b x c Where:  ε = molar absorptivity, the fraction of a specific wavelength of light absorbed by a given type of molecule  b = length of the light path through the solution  c = concentration of absorbing molecules 3. Unknown Solution: Au / As x Cs PERCENT TRANSMITTANCE (%T) the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I)  %T = It / I0 x 100 Where:  It = transmitted light thru sample  I0 = intensity of light striking the sample QUALITY ASSURANCE IN SPECTROPHOTOMETRY: a. Wavelength or photometric accuracy – assessed by using special glass-type optical filters Didymium glass: has a broad absorption peak around 600 nm Holmium oxide: multiple absorption peaks with a sharp peak occurring at 360 nm QUALITY ASSURANCE IN SPECTROPHOTOMETRY ABSORBANCE CHECK performed using glass filters or solutions that have known absorbance values for specific wavelength LINEARITY ability of a photometric system to yield a linear relationship between radiant power incident upon its detector and the concentration determined using optical filters or solutions that have known absorbance values for a given wavelength evaluated both slope and intercept STRAY LIGHT any light that impinges upon the detector that goes not originate from a polychromatic light source; evaluated by using special cutoff filters