Clinical Chemistry 1

CLINICAL CHEMISTRY 1 ANALYTIC TECHNIQUES ANALYTIC TECHNIQUES SPECTROMETRY LUMINESCENCE ELECTROANALYTIC METHODS CHROMATOGRAPHY Spectrophotometry Fluorescence Electrophoresis Gas Atomic Absorption Spectrometry Chemiluminescence Potentiometry Liquid Amperometry Thin Layer Mass Spectrometry SPECTROPHOTOMETRY 420m- - Bends the most 470 ー 는 580 620 ー 700 _ - Bends the least 一 个 Spectrophotometry – Instrument utilized to measure the concentration of the substance by measuring the light transmitted with respect to the light absorbed by the solution. SINGLE-BEAM SPECTROPHOTOMETER DOUBLE BEAM-SPECTROPHOTOMETER • Simplest type of absorption • The purpose of additional beam is to spectrophotometer correct for variation in light source intensity • Designed to make one measurement at a • Absorbance of the sample can be recorded time one specified wavelength directly as the electrical output of sample beam Beer’s Law • The concentration of the substance that is being measured is ______________________ Directly proportional to the absorbed light and inversely ________________________ proportional to the amount of transmitted light (%T) concentration 2 100 % A Absorbance e #" = sorptivity If - T ㉑ PARTS OF SPECTROPHOTOMER path 1. Light Source: Beam of light directly towards the monochromator and sample • Types of Light Source: o Continuum: Widely used in laboratory; Emits radiation that changes in intensity Tungsten Visible, IR Deuterium UV Xenon lamps UV o Line Source: Emits limited radiation and wavelength - Examples: Mercury and sodium vapor lamps (UV and Vis) and Hollow Cathode Lamp (AAS) 2. Entrance Slit: Minimizes stray light, preventing scattered light into monochromator 3. Monochromator: Selects a specific wavelength of light • Types of Monochromators o Glass filters - Simple, least expensive, and not precise - Filters produce monochromatic light constructiveinterference of waves ▪ Based on the principle of _________________________ ▪ Light waves enter 1st side of filter and are reflected on 2nd surface - Made by placing semi-transparent silver films on both sides of dielectric such as _________________________ Magnesium Fluoride o Diffraction Gratings - Commonly used and it has better resolution compared to prism - Made by parallel grooves Leslychrimatic light L - Special Type: Holographic gratings o Prisms - Wedge shaped pieces of sodium chloride, glass, or quartz 4. Exit Slit: Manipulate the width of light beam (bandpass) which results to a narrow fraction of spectrum to reach sample cuvette • ________________ Bandpass - The range of wavelength that is transmitted. 5. Sample cell: Contains the unknown solution to be measured Automated Icm : The path length of cuvette is ____; smaller path lengths used in _________. systems • Types of Cuvette: o Soft glass o Borosilicate glass o Alumina silica glass: Most commonly used (350nm to 2000nm) o Quartz: Measurement of unknown solution that requires Vis and UVs (NADH @ 340nm) • Sample cell with scratch optical surfaces scatter light should be discarded 6. Photodetector: convert the transmitted radiant energy into an equivalent amount of electrical energy. • Types of Photodetectors o Barrier-layer Cell/Photo Cell/Photovoltaic Cell - Least expensive, composed of film of light-sensitive material (Selenium on a plate of iron.) - Temperature sensitive - Photocells require no external voltage source but rely on internal electron transfer to produce a current in an external circuit. o Photomultiplier Tube - Most sensitive photodetector 2O0 - __________ times more sensitive than the phototube - Incident light strikes the coated cathode, emitting electrons. The electrons are attracted to a series of anodes, known as dynodes, each having a successively higher positive voltage. - Initial electron emission at the cathode triggers a multiple cascade of electrons within the PM tube itself. ㆁ o Photodiode - Less sensitive than PM tubes because of the lack of internal amplification - If incorporated in spectrophotometer as Photodetector Array (PDA), the grating is positioned after sample cuvette. o Phototube - It has photosensitive material that gives off electrons when light energy strikes it. External voltage - It requires an _____________________ for operation - Phototubes contain a negatively charged cathode and a positively charged anode enclosed in a glass case. 7. Readout Device: Displays the result of the detection system. • Types: Galvanometer, Light-emitting diode (LED) display, Ammeter 1. 2. 3. 4. Spectrophotometer Quality Assurance Absorption peak of 600nm Wavelength Accuracy : Didymium glass : oxide Multiple Absorption ul sharp peakof 360nm Holmium Absorbance Check : use glass Filters Linearity · Neutral Density Filters ; Dichromate solution Stray Light: any wavelength outside the band of interest. : ATOMIC ABSORPTION SPECTROPHOTOMETRY (AAS) Principle: • Measures concentration by detecting the absorption of electromagnetic radiation by atoms • Element is not excited but merely dissociated from its chemical bond → Ground state Measures: Not easily excited trace metals (Calcium and Magnesium) Components: • Light Source: Hollow cathode lamp • Chopper: Modulates the light source • Atomizer (Nebulizer/graphite furnace): Converts ions to atoms • Flame: _________; Disassociates chemical bond Internal standard is not needed Lanthanum or Strontium chloride may be added in the interference of phosphate Atomization Flameless AAS • Background correction is more needed on this type of atomic absorption spectrophotometry Electrothermal FLAME EMISSION SPECTROPHOTOMETRY (FES) Principle: Excitation of electrons from lower to higher energy state Light Source: Flame Cuvette: ___________ Flame Method: Indirect Internal Standard Method • Internal Standard: Lithium or Cesium Mechanism: Measures the light emitted by a single atom burned in a flame Measures: Sodium and Potassium Result: Light given off by the excited atoms is proportional to the concentration of the metal ions Less sensitive than AAS - TURBIDIMETRY AND NEPHELOMETRY TURBIDIMETRY amt Determines the ____________________ by a light blocked particulate matter in turbid solution . Depends on: • Specimen concentration • Particle Size • Turbidimetry is utilized in: - Antibiotic sensitivities - Coagulation analyzers - Quantify protein concentration in biologic fluids, such as urine and CSF NEPHELOMETRY amount of light scatteredby a Determines the __________________________ particulate matter suspended in a turbid solution Depends on: • Wavelength • Particle size Basic Components: • Light Source - Mercury-arc lamp - Tungsten-filament lamp - Light Emitting diode - Laser:____________________________ Light Amplification by Stimulated • Collimator Emission of Radiation • Monochromator • Sample Cuvette • Stray light trap • Photodetector: ______________________ Photomultiplier Tube (pmi) Utilized in Antigen-Antibody complexes (Diameter: 2501500 nm) - Wavelengths used are: 320nm-650nm 一 ELECTROPHORESIS, POTENTIOMETRY, AMPEROMETRY 1. Electrophoresis • Migration of charged particles in an electric field 8 cation : t Cathode : FACTORS AFFECTING RATE OF MIGRATION Anode : Anion : 1. Net charge of molecule molecule and 2. size molecular size shape of : separated by to protein cellulose Acetate Electri 3. field strength electrical charge ; It does notbird : separatesby : Gel medium size Agarose 4. Nature of supporting basis of changed molecular Gel : Separates Polyacrylamide 5. Temperature of the process Q on O PRINCIPLE: • Measures electrical potential (voltage) between two electrodes. • Used in: - pH measurement - Ion-selective electrodes (Na⁺, K⁺, Cl⁻) ^ - Blood gas analyzers • The potential difference between reference and indicator electrode is proportional to ion concentration. MAIN PARTS AND FUNCTION: CO2 PART DESCRIPTION • Sensitive to specific ion. - Example: Glass electrode : severinghaus INDICATOR • Directly responds to changes in the activityelectroe ELECTRODE or concentration of the target analyte in the solution. 、 oH4202P REFERENCE ELECTRODE VOLTMETER • • • Provides constant stable potential. Example: Ag/AgCl electrode : pH 3 CO2 Measures potential difference ↳ covered w/ ↳ kCI I saturated Calomel Electrode - mercury 3. Amperometry : por , glucose, Cl , peroxidase • Measures _________ current Flowproduced by an oxidation reaction MASS SPECTROMETRY PRINCIPLE: Determines the components of a sample where the • Identifies compounds and determines: - Molecular weight - Structure - Isotopic composition • Utilization in: - Drug testing - Proteomics - Toxicology - Newborn screening Clark Electrode : por ADDITIONAL CONCEPT: 1. Sample is ionized. 2. Ions are separated based on mass-tocharge ratio (m/z). 3. Detector measures ions. 4. Produces mass spectrum. MAIN PARTS AND FUNCTION: PARTS DETECTOR • VACUUM SYSTEM DATA SYSTEM • DESCRIPTION Converts molecules into ions Example includes: - E lectron lonization - M ALDI - & AElectrosprayionization Separates ions based on m/z ratio Mass analyzer type includes: - Quadrupole - Time of Flight - Ion Trap Designed to detect ions and convert impact into electrical signal Prevents ion collision with air molecules. • • Produces mass spectrum Identifies compound • • ION SOURCE • • MASS ANALYZER QUALITY MANAGEMENT IN CLINICAL LABORATORY Quality Management System - Involves standardized framework of reliability in every aspect of institution’s operation - Comprises of competent staff, advanced equipment, and continuous quality improvement Laboratory must ensure accurate and reliable results, achieved mainly by: - Quality Assurance - Quality Control – Checks the: o Stability of the machine o Quality of the reagents o Check technical (operator) errors CHARACTERISTICS OF IDEAL QC MATERIAL 1. Similar to human sample 2. Stable for long period of time & inexpensive 3. No matrix effects/ known matrix effects 4. With known analyte concentrations 5. Convenient packaging Internke o o o o INTRALAB QUALITY CONTROL Involves analysis of control together with patient specimens Detects changes in performance between present and stable operation Detects both random and systematic errors Important for daily monitoring 6. Free of communicable diseases ㅣ Externdac o o o o _ o o CONDUCT OF TESTING Initial control limits are establishing by analyzing pool material in 20 consecutive runs and reevaluated periodically QC results are checked after each run using multi-rule quality control programs; Hardcopy of QC results must be maintained o o o o \ national External ssessment the are INTERLAB es QUALITY CONTROL Involves Proficiency Testing Programs periodically. Maintains long-term accuracy Used to determine the State-of-the-Art interlaboratory performance CAP Proficiency Program is the gold standard for clinical laboratory interlab quality control testing CONDUCT OF TESTING Series of unknown samples from reference laboratory sent to laboratory Analysis of the sample must be processed completely same with routine samples Results must not be shared with other laboratories during testing cycle Proficiency tests can be qualitative or quantitative TYPES OF VARIATIONS/ERRORS Variations - Errors encountered in collection, preparation, and measurement of samples, including transcription and releasing of laboratory results. - Difference between test and reference method results RANDOM ERROR SYSTEMATIC ERROR - Present in all measurements; Positive or Negative - Can be result of many factors including: ▪ Instrument condition like temperature fluctuations ▪ Operator (Improper mixing of sample and reagent and Pipetting error) ▪ Environmental variation (electrical fluctuations) - Varies from sample to sample - Error that influences observations consistently in one direction - Detected as either positive or negative bias - Can be further subdivided into: ▪ Constant Error ▪ Proportional/Percent/Slope Error - Can be result of many factors including: ▪ Deterioration of reagents and control materials QUALITY CONTROL CHARTS Used to observe results or data of control materials over time to ensure the reliability of the analytical method 1. Gaussian Curve (Bell-Shaped Curve) o Occurs when data set can be accurately described by the SD and 10 99- mean o Obtained by plotting values from multiple analysis of sample o Focuses on the distribution of errors from analytical method rather than the values from healthy or patient 100 population o Total area under the curve is 1.0 or 100% EP = ∞ ofs 95 98 99 100 101 102 10 억 2. Youden/Twin Plot ー : f I o Use to compare results on high and low control serum from different laboratories o Categorizing of Errors: - ________________ Proportional Error – Points falling from the center and on the 45 degrees line Constant Error – Points - _________________ falling from center but not on 45 degrees line 3. Cumulative Sum Graph (CUSUM) o Calculates the difference between quality control results and target means V : mask o Common Method: ___________ - Utilized a sideways v-shaped to detect small sustained process shifts o Identifies consistent bias and requires computer implementation o Results are out of control when slope is beyond 45 degrees or a ± 2.7 SD. 4. Shewhart Levey-Jennings Chart o Graphic representation of acceptable limit errors in the results of the method used o Easily identifies random and systematic errors o Application of multiple rules in accepting and rejecting of controls ERRORS ON L-J CHART Outliers o ____________ - Values that are highly deviating - Can be caused by systematic errors or random errors Trend o ____________ - Values of the control that is consistently increasing or φ consecutive days decreasing for ___ - Main cause: Deterioration of ____________________ reagent o ____________ - Values of the control snift that distribute themselves on one side φ consecutive days for ____ - Main Cause: Improper ____________________ the instrument calibration of 5. Westgard QC Multi-Rules During the next 20 working days, the following QC results were obtained. LAB ACTIVITY The following Level 2 glucose quality control results (mg/dL) were obtained during instrument validation. Run 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ε 음 않 N F ( - - 邏 . ー . ー . - . . lonso = . . . ー - . . - - . . - X 20이 10428f 2 ) x * x * QC Result ー G5 101 0 9023 99 1. OS 1 102 s 100 0 OS P as 102 98 2 O5 101 99 100 102 98 101 100 99 101 aozs 0 93 100 -0.03 .0023 0 _ 4 98 2 05 . 2029 102 1 98 3 8023 _ 1 102 s 1 O5 99 ー 0 0023 00 s 100 _ . 9023 0 101 ogs : p 00 . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 QC Result 100 101 99 100 102 103 104 105 102 99 98 97 95 99 101 100 103 104 100 99 100.05 Iis I 2S 1 .9 3 s = 路 NORMAL GLUCOSE CONTROL 1 E 05 3 Run =1 35 . 41 = - 98 . 44 92 23 - - . 93 82 - . : mgldu · + 87 2102 . ① 101 46+ | · . · 10003X ..· 4 98 k - 0 I 23 297 "95. 82-3 - . 1 2 3 4 5 6 7 8 Run 9 10 (Day 11 12 X 13 14 15 16 17 18 19 101 20 , 44 10287 104 28 . PERFORMANCE IMPROVEMENT PROGRAMS 1. SIX SIGMA (6σ) o Goal is to improve the process by elimination of errors or variations o Indicators of this program is to improved: - Performance - Quality - Bottom line - Customer satisfaction - Employee satisfaction o Defects are anything which doesn’t meet the customer’s satisfaction or requirements such as: - Delay in Reporting in laboratory Result - E - Q vality Control Problem Mors Sigma Level 6 5 4 3 2 1 - DPMO 3.4 200 6,210 66,800 308,000 690,000 Efficiency Performance 99 9997 f world class 99 9800 y Excellent Good . . % . aa . 3790 % Good 93.3200% 69 2000%/ Poor % . 3 ) 0000 % Poor HOW DOES SIX-SIGMA SOLVE THE PROBLEM? Define the project goal or other deliverable which is a critical part for a quality Measure the baseline performance and related variables Analyze the data using statistics and graphs to identify and quantify root cause Improve performance by developing and implementing a solution Control factors related to the improvement, verify impact, validate benefits, and monitor over time 2. LEAN SYSTEM o System for reducing non-valued activities in production or manufacturing system - D efects - O verproduction - Waiting - Non-utilized talent - Transport - I nventory - M otion - E xtra processing o Utilizes the 5S and PDCA systems 5S PDCA Sort Plan Set in Order Do Shine Check Standardize Act Sustain 3. LEAN SIX SIGMA TAKE NOTE!! 1. To derive reliable estimates of reference intervals, at least ______ α⑳ individuals should be tested in each of the age and gender categories 2. Only ____ ⑳ reference individuals need to be sample for analysis on test instrument if laboratorian determines that the instrument and subject population are similar in the package insert of the manufacturer 3. The manufacturer’s reference limits may be accepted provided not more than 10% of the tested subjects fall outside the original reported limits; if greater that 10%, an additional 20 or more should be analyzed. 4. Reference values vary slightly depending the method and specimen type