Chapter 4: Systems of Measurement, Laboratory Equipment, and Reagents PDF

CHAPTER 4 Systems of Measurement, Laboratory Equipment and Reagents  Adapted from Linne & Ringsrud's Clinical Laboratory Science, 5th Edition The Basics and Routine Techniques By Mary Louise Turgeon, EdD, MT(ASCP), CLS(NCA) INTRO  If lab results are to be use...

CHAPTER 4 Systems of Measurement, Laboratory Equipment and Reagents  Adapted from Linne & Ringsrud's Clinical Laboratory Science, 5th Edition The Basics and Routine Techniques By Mary Louise Turgeon, EdD, MT(ASCP), CLS(NCA) INTRO  If lab results are to be useful in patient care they must be accurate.  The use of high quality analytical methods and instrumentation is essential to laboratory work.  The MT must be fully competent at using these instruments and methods. SYSTEMS OF MEASUREMENT  The ability to measure accurately is the keystone of the scientific method – MTs must therefore have a working knowledge of measuring systems and units of measurement. METRIC SYSTEM AND SI UNITS Measurement Unit Name Abbreviation Length Meter m Mass Kilogram Kg Time Seconds s Amount of substance Moles mol Electrical current Amperes A Luminous intensity Kelvin K Temperature candela cd PREFIXES OF SI UNITS Prefix Symbol Factor Decimal Tera T 1012 1,000,000,000,000 Mega M 106 1,000,000 Kilo k 103 1000 Deka da 101 10 Centi c 10-1 0.01 Milli m 10-3 0.001 Micro  10-6 0.000 001 Nano n 10-9 0.000 000 001 Femto f 10-18 0.000 000 000 000 000 0001 LAB PLASTICWARE AND GLASSWARE  The advantages of plasticware are its affordability and durability; the disadvantages include leakage of constituents into solutions, permeability to water vapor, absorption of analtyes.  Glassware is used in all departments and is preferred above plasticware inmost cases of analysis.  Different types of glass are used for different purposes.  Glassware and plasticware in the lab include; pipettes, beakers, measuring BALANCES AND CENTRIFUGE  Balances are used to measure precise masses or weights for reagent preparation and standard solutions. See procedure 4-2  Centrifuges are used to separate suspensions through the application of high gravitational force by rapid rotation. LAB REAGENT WATER  Water is one of the most important and frequently used reagents in the lab to prepare reagents, reconstitute lypophilized materials and dilution of samples and thus must be free from interfering substances.  Type I Reagent Water- this is the most pure and is used to prepare standard solutions, controls, buffers and in high sensitivity quantitative procedures. This cannot be stored and must be used immediately after it is produced.  Type II Reagent Water- this is used for most procedures that do not require type I water. It is less pure than type I water.  Type III Reagent Water- this is less pure and closer to tap water. This can be used as a LAB REAGENT WATER  Water must be monitored at regular intervals to evaluate the performance of the purification system.  The parameters tested should include: o Microbial monitoring o Resistivity o pH o Pyrogens o Silica o Organic contaminants  Methods of water purification include distillation, deionization, reverse osmosis, ultrafiltration, UV oxidation and sterilization, and ozone.  REAGENTS USED IN LAB ASSAYS.  A reagent is defined as any substance employed to produce a chemical reaction.  Chemicals are produces in various grades of purity as follows: o Analytical reagent (AR) grade- highest degree of purity o Chemically pure (CP) grade- less pure than AR o USP and NF grade- les pure than CP o Technical or commercial grade- not used in med lab.  Reagents must be store according to manufacturers instructions.  CHAPTER 6 Measurement Techniques in the Clinical Laboratory INTRO  Analytical techniques and instrumentation provide the foundation for all measurements made in the clinical lab.  Most measurement techniques fall into four basic categories: o Spectrometry o Luminescence o Electroanalytical methods o Chromatography  Spectrometry  Spectrophotometry is the use of light of specific wavelengths to measure the concentration of substances.  The spectrophotometer passes light of wavelengths of the visible or invisible spectrum through a sample and reads the amount of light absorbed by the sample.  The absorbance is directly proportional to the concentration of the analyte in the sample.(Beer-Lambert’s law.)  The equation for Beer-Lambert’s law is, subsequently,  A standard curve is plotted using the concentration vs the absorbance of the standard solutions. This is then used to find the concentration of patient samples.  This principle is applied to methods such as reflectance spectrophotometry, fluorescence spectrophotometry. DIAGRAMS NEPHELOMETRY AND TURBIDOMETRY  Nephelometry is the measurement of light that has been scattered.  Turbidimetry is the measurement of the loss of light due to scatter caused by particles in suspension.  This analysis is done with light of wavelength of 840nm.  The amount of light scattered is directly proportional to the concentration of the analyte. DIAGRAMS FLOW CYTOMETRY  This principle uses laser technology to analyze the samples.  First, the cells are stained with compounds (dyes) that will fluoresce when hit by light of sufficient intensity and a particular wavelength.  These compounds stain particular organelles and cell components and include FITC, TRITC, XRITC.  The stained cells are then passed through a laser beam in single file and the dyes fluoresce.  Each cell is analyzed for fluorescence at a rate of 500-5000 cells per second.  The number of cells positive for the analyte being studied is recorded as either a percentage or concentration. DIAGRAMS IMMUNOASSAYS  Immunoassays employ antibody-antigen reactions to determine the concentration of analytes in a sample.  Samples are placed in receptacles containing antibodies and treated with reagents that either contain antibodies or regulate their reactions while allowing there detection.  The compounds that allow the detection of the analytes are called labels and include enzymes, fluorescenes, chemiluminescent, and radio isotopes.  The intensity of the detection signal is proportional to the concentration of the analyte. DIAGRAMS ELECTROCHEMICAL METHODS  Potentiometry – measures the potential difference or voltage between two electrodes in a solution due to interactions with the analytes; the amount of voltage produced is directly proportional to the concentration of the analyte.  Electrophoresis is the movement of molecules in a electrical field; separation of the compounds are achieved due to differences in the speed of movement of the analytes.  Chromatography is the separation and detection of solutes in dissolved in a common solvent; the two main categories are gas and liquid.