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INSTRUMENT AND QUALITY CONTROL
Medical Technology (BSMT1)
Emilio Aguinaldo College
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Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda
WEEK 1: INTRODUCTION TO IQC
REAGENTS pre-packaged and readily available “kit” form. Little need for reagent preparation
Chemicals Analytic reagent (AR) Ultrapure, chemically pure (CP) United States Pharmacopeia (USP) National Formulary (NF) Technical or commercial grade Analytic reagent (AR) suitable for use in most analytic laboratory procedures the chemical meets the specifications of the American Chemical Society Ultrapure, chemically pure (CP) used in specific procedures such as chromatography, atomic absorption, immunoassays, molecular diagnostics, standardization, or other techniques that require extremely pure chemicals not recommended for clinical and analytic laboratory use United States Pharmacopeia (USP) and National Formulary (NF) used to manufacture drugs may be pure enough for use in most chemical procedures; however, it should be recognized that the purity standards are not based on the needs of the laboratory Technical or commercial grade used primarily in manufacturing and should never be used in the clinical laboratory
Organic Reagents Practical grade – with some impurities CP – purity level of reagent grade chemicals Spectroscopic (spectrally pure) and Chromatographic grade organic reagents – purity levels attained by their respective procedures Reagent grade (ACS) – contain impurities below certain levels established by the ACS
REFERENCE MATERIALS
Primary Standard a highly purified chemical that can be measured directly to produce a substance of exact known concentration and purity ACS purity tolerances for primary standards are 100 +- 0% National Institute of Standards and Technology (NIST) certified standard reference materials (SRMs) are used instead of ACS primary standard materials. developed certified reference materials/SRMs for use in clinical chemistry laboratories often used to verify calibration or accuracy/bias assessments there are SRMs for a number of routine analytes, hormones, drugs, and blood gases, with others being added.
Secondary Standard a substance of lower purity with concentration determined by comparison with a primary standard depends not only on its composition, which cannot be directly determined, but also on the analytic reference method
WATER SPECIFICATIONS “Water is the most frequently used reagent in the laboratory” Water solely purified by distillation results in distilled water Water purified by ion exchange produces deionized water Reverse osmosis, which pumps water across a semipermeable membrane, produces RO water Water can also be purified by ultrafiltration, ultraviolet light, sterilization, or ozone treatment
REAGENT GRADE WATER
Clinical and Laboratory Standard Institute Classified into 6 categories based on the specifications needed for its use rather than the method of purification or preparation
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda Classification clinical laboratory reagent water (CLRW), special reagent water (SRW) – HPLC, Molecular diagnostics and spectrophotometric techniques instrument feed water, water supplied by method manufacturer, autoclave and wash water, and commercially bottled purified water “water parameters include at least microbiological count, pH, resistivity, silicate, particulate matter, and organics”
CONVENTIONAL CLASSIFICATION Type 1 most stringent requirements and generally suitable for routine laboratory use used for test methods requiring minimum interference, such as trace metal, iron, and enzyme analyses Type 2 acceptable for most analytic requirements, including reagent, quality control, and standard preparation Type 3 acceptable for glassware washing but not for analysis or reagent preparation.
TECHNIQUES OF WATER PURIFICATION
Pre-filtration remove particulate matter filtration cartridges are composed of: glass; cotton; activated charcoal, which removes organic materials and chlorine o glass or cotton filter – used for hard water containing calcium, iron, and other dissolved elements submicron filter (<- 0 mm), which remove any substances larger than the filter’s pores, including bacteria o may be better suited after distillation, deionization, or reverse osmosis treatment Distillation water is boiled and vaporized the vapor rises and enters into the coil of a condenser, a glass tube that contains a glass coil cool water surrounds this condensing coil, lowering the temperature of the water vapor
the water vapor returns to a liquid state, which is then collected.
Ion-exchange it is neither pure nor sterile deionized water is purified from previously treated water, such as prefiltered or distilled water uses either an anion or a cation exchange resins=n, followed by replacement of the removed particles with hydroxyl or hydrogen ions
B. Ion Exchange (or) Demineralization (or) Deionization Process mineral ions are removed physical process which uses specifically manufactured ion exchange resins which bind to and filter the minerals salts from water
Reverse osmosis is a process that uses pressure to force water through a semipermeable membrane, producing water that reflects a filtered product of the original water
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda WEEK 2: INSTRUMENTS FOR MEASUREMENT
Glassware use in the following categories: - should consist of high terminal borosilicate content and aluminosilicate glass and meet the class A tolerances recommended by NIST. - The manufacturers are the best source of information about the uses, limitation, accuracy specification of the diff. types of glassware.
- plastic ware is beginning to replace glassware
- the unique resistance to corrosion and breakage as well as varying flexibility has made the plastic ware more appealing.
- relatability inexpensive it allows the most items to be completely disposable after each use. (Minimize washing of the different glassware.)
- the manufacture is the best...
- the major of resins frequently use in clinical lab chemistry are the:
PIPETTES
Made of glass or plastic Used to transfer liquids May be reusable or disposable Automatic pipettes – electronically operated
Pipette Classification Design To Contain (TC) To Deliver (TD) Drainage characteristics Blow-out Self-draining Type Measuring/graduated Serologic Mohr Bacteriologic Ball, Kolmer/Kahn Micropipettes (< 1 mL) Transfer Volumetric Ostwald-Folin Pasteur pipette Automatic macro/micro pipettes According to Design:
- “To Contain” Pipette Also called “rinsed-out pipette” They must be rinsed out with appropriate solvent after the initial liquid has been drained from the pipette
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda
Holds a particular volume but does not dispense that exact volume Ex. Sahli-Hellige pipette, Lang-Levy pipette, RBC and WBC pipettes, Kirk and Overflow pipette. 2. “To Deliver” Pipette Dispensed the volume indicated Should not be blown out Tip is placed against the side of the container and must not touch the liquid in it Ex. Mohr, serological, volumetric pipette According to Drainage characteristics:
- “Blow out” pipette With etched ring/2 continuous rings very close together The last drop of liquid should be expelled into the receiving vessel
- Self-draining pipette No etching rings Drained by gravity According to Type: Measuring/ graduated pipette divided into: dispense several different volumes
- Serologic pipette Calibration marks to the tip To blow out pipette/TD the graduation marks continue to the tip
- Mohr pipette
Does not have graduations to the tip
Self-draining pipette / TD
the graduations on these always end before the
tip
SPECIFICATIONS ON A MEASURING PIPETTE
Printed on the neck of the pipette are the
specifications that indicate:
the maximum volume of liquid that can be
transferred
the size of the divisions on the pipette
the temperature at which calibrations were made
if the pipette is a “to deliver” (TD) or “to contain”
(TC) pipette.
According to Transfer pipette:
- dispense one volume without further subdivisions
- Volumetric pipette With open ended bulb which holds the bulk of the liquid Self-draining/TD
Greatest degree of accuracy and precision and should be used when diluting standards, calibrators or QC material 2. Ostwald-Folin pipette
- Used with biologic fluids having a viscosity that is greater than water
- Blow out/TD
- Pasteur pipette
- Does not have calibrations
- Used to transfer solutions/biologic fluids without considering exact volume
- Not used for quantitative analytical techniques
- Mechanical/Automatic Micropipettes
- Deliver volume from 1-1000 uL Air displacement pipette Piston operated device Relies on piston for suction creation to draw the sample into a disposable tip. With disposable polypropylene tip Positive displacement pipette Operate by moving the piston in the pipette tip/barrel, much like a hypodermic syringe Used for reagents that reacts with plastic With siliconized glass tip (no need to replace tip Dispenser/dilutor pipette Obtain the liquid from a common reservoir and dispense it repeatedly Combines sampling and dispensing functions HELPFUL HINTS FOR ACCURATE PRECISE MEASUREMENTS USING MECHANICAL PIPETTES
- Pipettes should be stored in an upright position
- Prewet the pipette tip
- Work at temperature equilibrium
- Examine the tip before dispensing sample
- Use standard mode pipetting
- Pause consistently after aspiration
- Pull the pipette straight out
- Minimize handling of the pipette and tip
- Immerse the tip to the proper depth
- Use the correct pipette tip
- Use consistent plunger pressure and speed
- Do not use adjustable pipettes outside recommended ranges.
- Calibrate pipettes every six months, more frequently if the lab is clinical in nature.
- Prime the piston by depressing the plunger a few times prior to using the pipette for the first time each day of use.
- Leaks in the seal will adversely affect the reliability
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda International Organization for Standards (ISO) adopted by laboratories for standardization develops standards of practice, definitions and guidelines that provide for more uniform terminology and less confusion recommended that analytes be reported using moles of solute per volume of solution (substance concentration) moles/L
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda Beer’s Law: Absorbance = (ε) (l) (c) ε = molar absorptivity (constant for each type of molecule) l = length of light path through the solution (cuvet) c = concentration of the molecule absorbing the light HOW TO DETERMINE CONCENTRATION OF THE ANALYTE Three methods: proportionality graphing beer’s equation PROPORTIONALITY
A solution with a concentration of 0 is measured to have an absorbance of 0. Another solution of the same chemical is measured under the same conditions and has an absorbance of 0. What is its concentration? (0) / C2 = 0 / 0. C2 = (0 x 0) / 0. C2 = 0 GRAPHING Involves STANDARD SOLUTIONS
What is the concentration of a 1 cm (path length) sample that has an absorbance of 0?
BEER’S EQUATION
Absorbance = εlc
The molar absorptivity constant of a particular chemical is 1 L/mol·cm. What is the concentration of a solution made from this chemical that has an absorbance of 0 with a cell path length of 1? A = εbc 0 = 1 L/mol x 1 cm x C C = (0) / (1/M x 1) = 0 mol/L SPECTROPHOTOMETRY involves measurement of the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution. method how much a chemical substance absorbed light by measuring the intensity of light as a beam of light passes through a sample solution the basic principle each compound absorbed are transmits light over a certain range of wavelength measure the amount of known chemical substance is one of the useful methods of quantitative analysis in various fields of such as chemistry, physics, biochemistry, and etc. - It is the measurement of light intensity in a narrower wavelength - Photometric measurement – measurement of light intensity without consideration of wavelength PARTS OF SPECTROPHOTOMETER
- Light source
- Continuum source (emits radiation that changes in intensity)
- Line source (emits limited radiation and wavelength) Tungsten light bulb (most commonly used light source in the visible and near infrared ragion)
- Factors for choosing a light source: range, spectral distribution w/in the range, the source of radiant radiation, stability of the radiant energy and temperature
- Alternatives: Mercury arc (visible and UV) Deuterium lamp (UV) Hydrogen lamp (UV)
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda Xenon lamp (UV) Merst glower (IR) Globar/ silicon carbide (IR)
Entrance Slit
- Minimizes unwanted or stray light
- prevents the entrance of scattered light in monochromator system
- stray light – refers to any wavelength outside the bond transmitted by the monochromator. It causes absorbance error and strayed light limits the maximum absorbance that spectrophotometer can achieve.
Monochromator
- isolates specific or individual wavelength of light Differentiates spectrophotometer and photometer
- Kinds of monochromator Prisms (spectrophotometer) – used in a spectrophotometer with shape is a soft glass parts are sodium chloride and a narrow light focus on the prism is refracted as it enters the more dense glass. Can be rotated allowing only a desired wavelength to pass through and exit slits Diffraction gratings (spectrophotometer) - used in spectrophotometer. Most commonly used. Has a better resolution than prism. Made by cutting groves or slits into a aluminize surface of a flat twist of crown glass. The wavelengths are bend as hey pass a sharp corner. Filters (photometer) – used in photometer. It is simple and less expensive. Although not precise but very useful. Made by playsin??/resin semitransparent silver films on both side of a di electric such as magnesium fluoride. Produce monochromatic light based on principle of constructive interference of waves. The light waves enter the one side of the filter and are reflected at the second surface. Usually pass a wide bond of radiant energy and have a low transmittance of a selected wavelength.
Exit slit
- controls the width of light beam (bandpass)
- allows only a narrow fraction of a spectrum to reach the sample cuvet.
Cuvet
- also called absorption cell /analytical cell /sample cell
- holds the solution whose concentration is to be measured
- cuvet with scratches on their surface scattered light and should be discarded
- Kinds of cuvets: Alumina silica glass – the most commonly used. Quartz/plastic – used for measurement of solution requiring UV spectrum Borosilicate glass – for strong alkaline solution Soft glass – for strong acidic solution
Photodetector
- detects and converts transmitted light into a photoelectric energy
- detects the amount of light that passes through the sample in the cuvet
- Kinds of detector: barrier layer cell / photocell / photovoltaic cell – simplest detector, least expensive, and temperature sensitive. Composed of selenium on a plate of iron covered with transparent layer of silver. Requires no external voltage source, but utilized internal electron transfer for current reduction. Has a low internal resistance. Use in in filter photometer with a wide bandpass. phototube – contains cathodes and anodes and enclose in glass case. It has a photo sensitive material that gives up electrons when light energy strikes it. It requires external voltage for operation photomultiplier (PM) tube – the most common type. It measures visible and UV regions. Excellent sensitivity and rapid response. Detect and amplifies radiant energy and detects very low light level and low analyte concentration. Photodiode – it is not as sensitive as PM, but it is excellent linearity. Measures light
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda
Iontophoresis – migration of small charged ions Zone electrophoresis – migration of charged macromolecules
Factors affecting rate of migration - Net electric charge of the molecule - Size and shape of the molecule - Electric field strength - Nature of the supporting medium - Temperature of operation
Supporting media: - Paper electrophoresis - Starch gel – separates by surface charge and molecular size - Cellulose acetate – separates by molecular size (protein) - Agarose gel – neutral; separates by electrical charge; it does not bind protein - Polyacrylamide gel – neutral; separates on the basis of charge and molecular size; separates proteins into 20 fractions; used to study isoenzymes
Stains for Visualization of Fractions - Amido black - Ponceau S - Oil Red O - Sudan Black - Fat Red 7B - Coomassie Blue - Gold/Silver
Isoelectric Focusing - Separating molecules migrate through a pH gradient; uses a constant power. - Proteins move in the electric field until they reach a pH equal to their isoelectric point. - Ideal for separating proteins of identical sizes but different net charges (isoenzymes). - Supporting media: agarose gel, polyacrylamide gel and cellulose acetate
Advantages: - The ability to resolve mixture of proteins - To detect isoenzymes of CK and ALP in serum - To identify genetic variants of proteins such as alpha-1-antitrypsin - To detect CSF oligoclonal banding
Densitometry - Measures the absorbance of stain - Scan and quantitate electrophoretic pattern - Reads gel and cellulose acetate membrane
Capillary electrophoresis - Sample molecules are separated by electro- osmotic flow (EOF) - Positively charged ions in the specimen emerge early at the capillary outlet because the EOF and the ion movement are in the same direction - Negatively charged ions in the specimen move towards the capillary outlet but at a slower rate - Used nanoliter quantities of specimens - Uses: separation, quantitation and determination of molecular weights of proteins and peptides, analysis of PCR products, analysis of organic and inorganic substances and drugs
Western Blotting (immunoblotting) A method used to separate, detect and identify one or more proteins in a complex mixture Chromatography Involves the separation of soluble components in a solution by specific differences in physical- chemical characteristics of the different constituent
Three components thus form the basis of the chromatography technique. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.” Separated molecules
2 forms of chromatography
Professor: Mr. Apolonio Alecksandr T. Molina, RMT, MSMT Transcribed by: Myrell M. Retonda
Planar - Paper chromatography Fractionation of sugar and amino acids Sorbent – Whatman paper Thin Layer Chromatography (TLC) - Used for drug screening (semiquantitative screening test) - Sample components are identified by comparison with standards on the same plate - Sorbent – thin plastic plates impregnated with a layer of silica gel or alumina Column Gas chromatography Used for the separation of steroids, barbiturates, blood, alcohol and lipids Samples are introduced into the GC column using a hypodermic syringe or an automated sampler The specimens are vaporized and swept onto the column Useful for compounds that are naturally volatile or can be easily converted into a volatile form Mobile phase: nitrogen, helium, hydrogen and argon (Inert gases) Two types of GC - Gas Solid Chromatography (GSC) Differences in absorption at the solid phase surfaces - Gas Liquid Chromatography Separation occurs by differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase
Mass Spectrometry Before the compound can be detected and quantified by MS, it must be separated by GC Based on the fragmentation and ionization of molecules using suitable source of energy Measures compound based on charge-to- mass ratio Gold standard for drug testing Separation mechanisms Gel/Gel permeation/Gel Filtration/ Size exclusion/ Molecular sieve chromatography - Separates molecules based on differences in their size and shape
- As solutes travel through the gel, large molecules remain in the mobile phase are eluted rapidly from the column Hydrophilic gel (Gel filtration)
- For separation of enzymes, antibodies and proteins
- Examples: dextran and agarose
Hydrophobic gel (Gel permeation) - For separation of triglycerides and fatty acids - Example: sephadex Ion exchange chromatography Separation of nucleic acids and proteins depends primarily on the sign and ionic charge density For separation of amino acids, proteins and nucleic acids
Partition chromatography (liquid-liquid chromatography) Separation is based on relative solubility in an organic non-polar solvent and an aqueous / polar solvent. For separation of therapeutic drugs and their metabolites
Affinity chromatography Used to separate and prepare larger quantities of proteins and antibodies for study For separation of lipoproteins, CHO and HbA1c
Adsorption Chromatography (liquid-solid chromatography) Separation is based on the differences (competition) between the adsorption and desorption of solutes at the surface of a solid particle. Electrochemistry Techniques The measurement of current or voltage generated by the activity of a specific ion
Potentiometry - Measures of differences in voltage (potential) at a constant rate - Useful for measuring blood pH - Nernst Equation - Reference electrode Saturated calomel (external)
INSTRUMENT AND QUALITY CONTROL
Course: Medical Technology (BSMT1)
University: Emilio Aguinaldo College
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