CHAPTER 1.pdf

CLINICAL CHEMISTRY CHAPTER 1 BASIC PRINCIPLES AND PRACTICES OF CLINICAL CHEMISTRY CLINICAL CHEMISTRY CHAPTER 1 Clinical Chemistry o The primary purpose of a clinical chemistry laboratory is to perform analytic procedures that yield accurate and precise...

CLINICAL CHEMISTRY CHAPTER 1 BASIC PRINCIPLES AND PRACTICES OF CLINICAL CHEMISTRY CLINICAL CHEMISTRY CHAPTER 1 Clinical Chemistry o The primary purpose of a clinical chemistry laboratory is to perform analytic procedures that yield accurate and precise information, aiding in patient diagnosis and treatment. o The achievement of reliable results requires that the clinical laboratorian be able to correctly use basic supplies and equipment and possess an understanding of fundamental concepts critical to any analytic procedure. CLINICAL CHEMISTRY CHAPTER 1 Units of Measure o Any meaningful quantitative laboratory result consists of two components: the first component represents the number related to the actual test value, and the second is a label identifying the units. o The unit defines the physical quantity or dimension, such as mass, length, time, or volume. There are a few laboratory tests that do not have units, but whenever possible, units should be used. CLINICAL CHEMISTRY CHAPTER 1 Units of Measure o The Système International d’Unités (SI) was adopted in 1960. It is preferred in scientific literature and clinical laboratories and is the only system employed in many countries. o This system was devised to provide the global scientific community with a uniform method of describing physical quantities. The SI system units (referred to as SI units) are based on the metric system. o Several subclassifications exist within the SI system, one of which is the basic unit. There are seven basic units: CLINICAL CHEMISTRY CHAPTER 1 Base Quantity Name Length Meter Mass Kilogram Time Second Electric Current Ampere Thermodynamic Kelvin temperature Amount of substance Mole Luminous intensity Candela CLINICAL CHEMISTRY CHAPTER 1 Units of Measure o Derived units are another subclassification of the SI system. o A derived unit is a mathematical function describing one of the basic units. o Example: meters per second [m/s] (velocity) o The SI system uses standard prefixes to indicate a decimal fraction or multiples of that basic unit. CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 Units of Measure o Reporting of laboratory results is often expressed in terms of substance concentration (e.g., moles) or the mass of a substance (e.g., mg/dL, g/dL, g/L, mmol/L, and IU) rather than in SI units. o As with other areas of industry, the laboratory and the rest of medicine are moving toward adopting universal standards promoted by the International Organization for Standardization, often referred to as ISO. o Many national initiatives have recommended common units for laboratory test results, but none have been widely adopted. As with any transition, the clinical laboratorian should be familiar with all the terms currently used in their field and how to convert these to SI units. CLINICAL CHEMISTRY CHAPTER 1 Reagents o Most instrument manufacturers make the reagents in a ready-to- use form or “kit” in which all necessary reagents and respective storage containers are prepackaged as a unit, requiring only the addition of water or buffer for reconstitution. o A heightened awareness of the hazards of certain chemicals and the numerous regulatory agency requirements has caused clinical chemistry laboratories to eliminate massive stocks of chemicals and opt instead for the ease of using prepared reagents. o Periodically, the laboratorian may still need to prepare reagents or solutions, especially in hospital laboratories involved in research and development, biotechnology applications, specialized analyses, or method validation. CLINICAL CHEMISTRY CHAPTER 1 Chemicals o Analytic reagent (AR) or reagent grade ▪ Chemicals with AR designation are suitable for use in most analytic laboratory procedures ▪ A committee of the American Chemical Society (ACS) established specifications for AR grade chemicals, and chemical manufacturers must either meet or exceed these requirements. ▪ The labels on reagents should clearly state the actual impurities for each chemical lot or list the maximum allowable impurities. The label should also include one of the following designations: AR or ACS or For laboratory use or ACS Standard-Grade Reference Materials. CLINICAL CHEMISTRY CHAPTER 1 Chemicals o Ultrapure chemicals ▪ Ultrapure chemicals have additional purification steps for use in specific procedures such as chromatography, immunoassays, molecular diagnostics, standardization, or other techniques that require extremely pure chemicals. ▪ These reagents may have designations of HPLC (high- performance liquid chromatography) or chromatographic on their labels. CLINICAL CHEMISTRY CHAPTER 1 Chemicals o United States Pharmacopeia (USP), National Formulary (NF) ▪ Because USP- and NF-grade chemicals are used to manufacture drugs, the limitations established for this group of chemicals are based only on the criterion of not being injurious to individuals. ▪ Chemicals in this group may be pure enough for use in most chemical procedures, but the purity standards they meet are not based on the needs of the laboratory and may or may not meet all assay requirements. CLINICAL CHEMISTRY CHAPTER 1 Chemicals o Chemically Pure ▪ It is not recommended that clinical laboratories use these chemicals for reagent preparation unless further purification or a reagent blank is included. o Technical or Commercial Grade ▪ Technical or commercial grade reagents are used primarily in manufacturing and should never be used in the clinical laboratory. CLINICAL CHEMISTRY CHAPTER 1 Chemicals o Organic reagents ▪ also have varying grades of purity that differ from those used to classify inorganic reagents. ✓ CP – approaches the purity level of reagent-grade chemicals; ✓ Spectroscopic (spectrally pure) and chromatographic grade organic reagents ✓ Reagent grade (ACS), which is certified to contain impurities below established ACS levels CLINICAL CHEMISTRY CHAPTER 1 Chemicals o Reference Materials ▪ Primary Standard – highly purified chemical that can be measured directly to have an exact known concentration and purity. ▪ The ACS has purity tolerances for primary standards; because most biologic constituents are unavailable within these tolerance limitations, the National Institute of Standards and Technology (NIST) has certified standard reference materials (SRMs) that are used in place of ACS primary standard materials. CLINICAL CHEMISTRY CHAPTER 1 Chemicals o Reference Materials ▪ These SRMs are assigned a value after analysis using state-of-the- art methods and equipment. The chemical composition of these substances is then certified; however, they may not have the purity of a primary standard. ▪ Because each substance has been characterized for certain chemical or physical properties, it can be used in place of an ACS primary standard in clinical work and is often used to verify calibration or accuracy/bias assessments. ▪ Standard reference materials are used for linearity studies to determine the relationship between the standard’s concentration and the instrument result. Linearity studies are required when a new test or new test methodology is introduced. CLINICAL CHEMISTRY CHAPTER 1 Water o Water Specifications ▪ Water is the most frequently used reagent in the laboratory. Tap water is unsuitable for laboratory applications. ▪ Most procedures, including reagent and control preparation, require water that has been substantially purified, known as reagent-grade water. ▪ There are various water purification methods including distillation, ion exchange, reverse osmosis, ultrafiltration, ultraviolet light, sterilization, and ozone treatment. CLINICAL CHEMISTRY CHAPTER 1 Water o Reagent Grade Water ✓ Clinical Laboratory Regent Water (CLRW) ✓ Special Reagent Water (SRW) ✓ Instrument Feed Water ✓ Water Supplied by Method Manufacturer ✓ Autoclave and Wash Water ✓ Commercially Bottled Purified Water ▪ Water quality testing routinely includes monitoring microbial colony-forming units/mL and may also include other parameters. CLINICAL CHEMISTRY CHAPTER 1 Water o Distilled Water ▪ Distilled water - been purified to remove almost all organic materials, using a technique of distillation where water is boiled and vaporized. ▪ Many impurities do not rise in the water vapor and will remain in the boiling apparatus so that the water collected after condensation has less contamination. ▪ Water may be distilled more than once, with each distillation cycle removing additional impurities. ▪ Ultrafiltration and nanofiltration, like distillation, are excellent in removing particulate matter, microorganisms, and any pyrogens or endotoxins CLINICAL CHEMISTRY CHAPTER 1 Water o Deionized water ▪ Deionized water has some or all ions removed, although organic material may still be present, so it is neither pure nor sterile. ▪ Generally, deionized water is purified from previously treated water, such as prefiltered or distilled water. ▪ Deionized water is produced using either an anion- or a cation- exchange resin, followed by replacement of the removed ions with hydroxyl or hydrogen ions. ▪ A combination of several ion-exchange resins will produce different grades of deionized water. CLINICAL CHEMISTRY CHAPTER 1 Water o Reverse osmosis ▪ Reverse osmosis is a process that uses pressure to force water through a semipermeable membrane, producing a filtered product. ▪ Reverse osmosis may be used for the pretreatment of water, however, it does not remove dissolved gases. CLINICAL CHEMISTRY CHAPTER 1 Water o Filtration ▪ Filtration can remove particulate matter from municipal water supplies before any additional treatments. ▪ Filtration cartridges can be composed of glass, cotton, or activated charcoal, which removes organic materials and chlorine. Some have submicron filters (≤0.2 µm), which remove any substances larger than the filter’s pores, including bacteria. ▪ The use of these filters depends on the quality of the municipal water and the other purification methods used. CLINICAL CHEMISTRY CHAPTER 1 Water o Filtration ▪ For example, hard water (containing calcium, iron, and other dissolved elements) may require prefiltration with a glass or cotton filter rather than activated charcoal or submicron filters, which quickly become clogged and are expensive to use. The submicron filter may be better suited after distillation, deionization, or reverse osmosis treatment. CLINICAL CHEMISTRY CHAPTER 1 Water o Filtration ▪ For example, hard water (containing calcium, iron, and other dissolved elements) may require prefiltration with a glass or cotton filter rather than activated charcoal or submicron filters, which quickly become clogged and are expensive to use. The submicron filter may be better suited after distillation, deionization, or reverse osmosis treatment. CLINICAL CHEMISTRY CHAPTER 1 Water o Filtration ▪ Ultraviolet oxidation, which removes some trace organic material or sterilization processes at specific wavelengths, can destroy bacteria when used as part of a system but may leave behind some residual products. This technique is often followed by other purification processes. CLINICAL CHEMISTRY CHAPTER 1 Water o Reagent-Grade Water ▪ Reagent-grade water can be obtained by initially filtering to remove particulate matter, followed by reverse osmosis, deionization, and a 0.2-µm filter or more restrictive filtration process. ▪ Autoclave wash water is acceptable for glassware washing but not for analysis or reagent preparation. ▪ SRW is used for specific techniques like the HPLC, molecular diagnostics, or mass spectrophotometry, which may require specific parameters for the analysis. ▪ All SRW should meet CLRW standards and, depending on the application, CLRW should be stored in a manner that reduces any chemical or bacterial contamination and for short periods. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o In clinical chemistry, substances found in biologic fluids, including serum, plasma, urine, and spinal fluid, are quantified. o A substance that is dissolved in a liquid is called a solute; a biologic solute is also known as an analyte. o The liquid in which the solute is dissolved—for example, a biologic fluid—is the solvent. o Together, solute and solvent represent a solution. o Any chemical or biologic solution can be described by its basic properties, including concentration, saturation, colligative properties, redox potential, conductivity, density, pH, and ionic strength. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Concentration ▪ The analyte concentration in solution can be expressed in many ways. Concentration is commonly expressed as percent solution, molarity, molality, or normality. These are non-SI units, however; the SI unit for the amount of a substance is the mole. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Concentration ▪ Percent solution – expressed as the amount of solute per 100 total units of solution ▪ Weight per weight (w/w), volume per volume (v/v), and weight per volume (w/v) ▪ Weight per weight (% w/w) refers to the number of grams of solute per 100 g of solution. ▪ Volume per volume (% v/v) is used for liquid solutes and gives the milliliters of solute in 100 mL of solution. ▪ For v/v solutions, it is recommended that grams per deciliter (g/dL) be used instead of % v/v. ▪ Weight per volume (% w/v) is the most commonly used percent solution in the clinical laboratory and is defined as the number of grams of solute in 100 mL of solution. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Molarity (M) ▪ Molarity (M) is expressed as the number of moles per 1 L of solution. ▪ One mole of a substance equals its gram molecular weight (gmw), so the customary units of molarity (M) are moles/liter. ▪ The SI representation for the traditional molar concentration is mole of solute per volume of solution, with the volume of the solution given in liters. ▪ The SI expression for concentration should be represented as moles per liter (mol/L), millimoles per liter (mmol/L), micromoles per liter (μmol/L), or nanomoles per liter (nmol/L). CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Molarity (M) ▪ The common concentration term molarity is not an SI unit for concentration. Molarity depends on volume, and any significant physical changes that influence volume, such as changes in temperature and pressure, will also influence molarity. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Molality (m) ▪ Molality (m) represents the amount of solute per 1 kg of solvent. ▪ Molality is sometimes confused with molarity; however, it can be easily distinguished because molality is always expressed in terms of moles per kilogram (weight per weight) and describes moles per 1000 g (1 kg) of solvent. ▪ Molality is not influenced by temperature or pressure because it is based on mass rather than volume. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Normality ▪ Normality is the least likely of the four concentration expressions to be encountered in clinical laboratories, but it is often used in chemical titrations and chemical reagent classification. ▪ It is defined as the number of gram equivalent weights per 1 L of solution. ▪ An equivalent weight is equal to the gmw of a substance divided by its valence. ▪ The valence is the number of units that can combine with or replace 1 mole of hydrogen ions for acids and hydroxyl ions for bases and the number of electrons exchanged in oxidation– reduction reactions. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Normality ▪ Normality is always equal to or greater than the molarity of the compound. ▪ Normality was previously used for reporting electrolyte values, expressed as milliequivalents per liter (mEq/L); however, this convention has been replaced with millimoles per liter (mmol/L). ▪ The College of American Pathologists (CAP) currently requires chloride to be reported in mmol/L. Because the four main electrolytes, Na+ , K+ , CO2 – (HCO3 – ), and Cl– , all have a valence of 1, the concentration reported will remain the same whether the unit is mEq/L or mmol/L. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Solution Saturation ▪ Solution saturation gives little specific information about the concentration of solutes in a solution. ▪ A solution is considered saturated when no more solvent can be dissolved in the solution. ▪ Temperature, as well as the presence of other ions, can influence the solubility constant for a solute in a given solution and thus affect the saturation. ▪ Routine terms in the clinical laboratory that describe the extent of saturation are dilute, concentrated, saturated, and supersaturated. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Solution Saturation ▪ Dilute solution - one in which there is relatively little solute or one that has a lower solute concentration per volume of solvent than the original, such as when making a dilution. ▪ Concentrated solution - has a large quantity of solute in solution. ▪ Saturated solution – a solution in which there is an excess of undissolved solute particles ▪ Supersaturated solution – has an even greater concentration of undissolved solute particles than a saturated solution of the same substance. Because of the greater concentration of solute particles, a supersaturated solution is thermodynamically unstable. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Colligative Properties ▪ Colligative properties are those properties related to the number of solute particles per solvent molecules, not on the type of particles present. The behavior of particles or solutes in solution demonstrates four properties: osmotic pressure, vapor pressure, freezing point, and boiling point. ▪ Osmotic pressure is the pressure that opposes osmosis when a solvent flows through a semipermeable membrane to establish equilibrium between compartments of differing concentration. ▪ Vapor pressure is the pressure exerted by the vapor when the liquid solvent is in equilibrium with the vapor. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Colligative Properties ▪ Freezing point is the temperature at which the first crystal (solid) of solvent forms in equilibrium with the solution. ▪ Boiling point is the temperature at which the vapor pressure of the solvent reaches atmospheric pressure (usually 1 atmosphere). CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Redox Potential ▪ Redox potential, or oxidation–reduction potential, is a measure of the ability of a solution to accept or donate electrons. ▪ Substances that donate electrons are called reducing agents; those that accept electrons are considered oxidizing agents. ▪ The mnemonic—LEO (lose electrons oxidized) the lion says GER (gain electrons reduced)—may prove useful when trying to recall the relationship between reducing/oxidizing agents. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o Conductivity ▪ The mnemonic—LEO (lose electrons oxidized) the lion says GER (gain electrons reduced)—may prove useful when trying to recall the relationship between reducing/oxidizing agents. ▪ Resistivity - the reciprocal of conductivity, is a measure of a substance’s resistance to the passage of electrical current. The primary application of resistivity in the clinical laboratory is for assessing the purity of water. ▪ Resistivity (resistance) is expressed as ohms and conductivity is expressed as ohms−1. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o pH and Buffers ▪ Buffers are weak acids or bases and their related salts that minimize changes in the hydrogen ion concentration. ▪ Hydrogen ion concentration is often expressed as pH. ▪ A lowercase p in front of certain letters or abbreviations operationally means the “negative logarithm of” or “inverse log of” that substance. ▪ In keeping with this convention, the term pH represents the negative or inverse log of the hydrogen ion concentration. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o pH and Buffers ▪ [H+ ] equals the concentration of hydrogen ions in moles per liter (M). The pH scale ranges from 0 to 14 and is a convenient way to express hydrogen ion concentration. CLINICAL CHEMISTRY CHAPTER 1 Solution Properties o pH and Buffers ▪ Ionic strength is another important aspect of buffers, particularly in separation techniques. Ionic strength is the concentration or activity of ions in a solution or buffer. ▪ Increasing ionic strength increases the ionic cloud surrounding a compound and decreases the rate of particle migration. It can also promote compound dissociation into ions effectively increasing the solubility of some salts, along with changes in current, which can also affect electrophoretic separation. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Heating Units ▪ Heat blocks and water baths are common heating units within the laboratory. The temperature of these heating units must be monitored daily when in use. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Heating Units ▪ All analytic reactions occur at an optimal temperature. Some laboratory procedures, such as enzyme determinations, require precise temperature control, whereas others work well over a wide range of temperatures. ▪ Reactions that are temperature dependent use some type of heating/cooling cell, heating/cooling block, or water/ice bath to provide the correct temperature environment. ▪ Laboratory refrigerator temperatures are often critical and need periodic verification. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Heating Units ▪ Thermometers can be an integral part of an instrument or need to be placed in the device for temperature maintenance and monitoring. ▪ Several types of temperature devices are currently used in the clinical laboratory, including liquid-in-glass and electronic (thermistor) devices. ▪ Regardless of which type is being used, all temperature-reading devices must be calibrated for accuracy. ▪ Liquid-in-glass thermometers use a colored liquid (red or other colored material), encased in plastic or glass, measuring temperatures between 20°C and 400°C. ▪ Visual inspection of the liquid in-glass thermometer should reveal a continuous line of liquid, free from separation or bubbles. If separation or bubbles are present, then replace the thermometer. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Heating Units ▪ Thermometers can be an integral part of an instrument or need to be placed in the device for temperature maintenance and monitoring. ▪ Several types of temperature devices are currently used in the clinical laboratory, including liquid-in-glass and electronic (thermistor) devices. ▪ Regardless of which type is being used, all temperature-reading devices must be calibrated for accuracy. ▪ Liquid-in-glass thermometers use a colored liquid (red or other colored material), encased in plastic or glass, measuring temperatures between 20°C and 400°C. ▪ Visual inspection of the liquid in-glass thermometer should reveal a continuous line of liquid, free from separation or bubbles. If separation or bubbles are present, then replace the thermometer. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Glassware and Plasticware ▪ Those that satisfy Class A ASTM precision criteria are stamped with the letter “A” on the glassware and are preferred for laboratory applications. ▪ Class B glassware generally have twice the tolerance limits of Class A, even if they appear identical, and are often found in student laboratories where durability is needed. ▪ Vessels holding or transferring liquid are designed either to contain (TC) or to deliver (TD) a specified volume. ▪ The major difference is that TC devices do not deliver the volume measured when the liquid is transferred into a container, whereas the TD designation means that the labware will deliver the amount measured. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Glassware and Plasticware ▪ Glassware used in the clinical laboratory usually fall into one of the following categories: Kimax/Pyrex (borosilicate), Corex (aluminosilicate), high silica, Vycor (acid and alkali resistant), low actinic (amber colored), or flint (soda lime) glass used for disposable material. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Glassware and Plasticware ▪ Plasticware is beginning to replace glassware in the laboratory setting; high resistance to corrosion and breakage, as well as varying flexibility, has made plasticware appealing. ▪ Relatively inexpensive, it allows most items to be completely disposable after each use. ▪ The major types of resins frequently used in the clinical chemistry laboratory are polystyrene, polyethylene, polypropylene, Tygon, Teflon, polycarbonate, and polyvinyl chloride. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ A volumetric flask is calibrated to hold one exact volume of liquid (TC). ▪ The flask has a round, lower portion with a flat bottom and a long, thin neck with an etched calibration line. ▪ Volumetric flasks are used to bring a given reagent to its final volume with the recommended diluent. ▪ When bringing the bottom of the meniscus to the calibration mark, a pipette should be used for adding the final drops of diluent to ensure maximum control is maintained and the calibration line is not missed. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Erlenmeyer flasks and Griffin beakers are designed to hold different volumes rather than one exact amount. ▪ Because Erlenmeyer flasks and Griffin beakers are often used in reagent preparation, flask size, chemical inertness, and thermal stability should be considered. ▪ The Erlenmeyer flask has a wide bottom that gradually evolves into a smaller, short neck. The Griffin beaker has a flat bottom, straight sides, and an opening as wide as the flat base, with a small spout in the lip. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Graduated cylinders are long, cylindrical tubes usually held upright by an octagonal or circular base. The cylinder has horizontal calibration marks and is used to measure volumes of liquids. Graduated cylinders do not have the accuracy of volumetric labware. The sizes routinely used are 10, 25, 50, 100, 500, 1000, and 2000 mL. ▪ All laboratory glassware used for critical measurements should be Class A whenever possible to maximize accuracy and precision and thus decrease calibration time CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Pipettes are a type of laboratory equipment used to transfer liquids; they may be reusable or disposable. Although pipettes may transfer any volume, they are usually used for volumes of 20 mL or less; larger volumes are usually transferred or dispensed using automated pipetting devices. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Similar to other laboratory equipment, pipettes are designed to contain (TC) or to deliver (TD) a particular volume of liquid. ▪ The major difference is the amount of liquid needed to wet the interior surface of the pipette and the amount of any residual liquid left in the pipette tip. ▪ Most manufacturers stamp TC or TD near the top of the pipette to alert the user as to the type of pipette. ▪ Like other TC-designated labware, a TC pipette holds or contains a particular volume but does not dispense that exact volume, whereas a TD pipette will dispense the volume indicated. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ When using either pipette, the tip must be immersed in the intended transfer liquid to a level that will allow the tip to remain in solution after the volume of liquid has entered the pipette—without touching the vessel walls. ▪ The pipette is held upright, not at an angle. ▪ Using a pipette bulb or similar device, a slight suction is applied to the opposite end until the liquid enters the pipette and the meniscus is brought above the desired graduation line. ▪ While the meniscus level is held in place, the pipette tip is raised slightly out of the solution and wiped with a laboratory tissue to remove any adhering liquid. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ The liquid is allowed to drain until the bottom of the meniscus touches the desired calibration mark. ▪ With the pipette held in a vertical position and the tip against the side of the receiving vessel, the pipette contents are allowed to drain into the vessel (e.g., test tube, cuvette, or flask). ▪ A blowout pipette has a continuous etched ring or two small, close, continuous rings located near the top of the pipette. This means that the last drop of liquid should be expelled into the receiving vessel. ▪ Without these markings, a pipette is self-draining, and the user allows the contents of the pipette to drain by gravity. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ The tip of the pipette should not be in contact with the accumulating fluid in the receiving vessel during drainage. With the exception of the Mohr pipette, the tip should remain in contact with the side of the vessel for several seconds after the liquid has drained. The pipette is then removed. CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Measuring or graduated pipettes are capable of dispensing several different volumes. Measuring pipettes are used to transfer reagents or make dilutions and can be used to repeatedly transfer a particular solution. ▪ The markings at the top of a measuring or graduated pipette indicate the volume(s) it is designed to dispense. Because the graduation lines located on the pipette may vary, the increments will be indicated on the top of each pipette. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ The subgroups of measuring or graduated pipettes are Mohr, serologic, and micropipettes. ▪ A Mohr pipette does not have graduations to the tip. It is a self- draining pipette, but the tip should not be allowed to touch the vessel while the pipette is draining. ▪ A serologic pipette has graduation marks to the tip and is generally a blowout pipette. ▪ A micropipette is a pipette with a total holding volume of less than 1 mL; it may be designed as either a Mohr or a serologic pipette. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Transfer pipettes are designed to dispense one volume without further subdivisions. Ostwald-Folin pipettes are used with biologic fluids having a viscosity greater than that of water. ▪ They are blowout pipettes, indicated by two etched, continuous rings at the top. ▪ The volumetric pipette is designed to dispense or transfer aqueous solutions and is always self-draining. The bulb-like enlargement in the pipette stem easily identifies the volumetric pipette. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ This type of pipette usually has the greatest degree of accuracy and precision and should be used when diluting standards, calibrators, or quality control material. ▪ They should only be used once prior to cleaning. ▪ Disposable transfer pipettes may or may not have calibration marks and are used to transfer solutions or biologic fluids without consideration of a specific volume. These pipettes should not be used in any quantitative analytic techniques. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ The automatic pipette is the most routinely used pipette in today’s clinical chemistry laboratory. ▪ Automatic pipettes come in a variety of types including fixed volume, variable volume, and multichannel. ▪ The term automatic, as used here, implies that the mechanism that draws up and dispenses the liquid is an integral part of the pipette. ▪ It may be a fully automated/self-operating, semiautomatic, or completely manually operated device. CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Automatic and semiautomatic pipettes have many advantages, including safety, stability, ease of use, increased precision, the ability to save time, and less cleaning required because the pipette tips are disposable. ▪ A pipette associated with only one volume is termed a fixed volume, and models able to select different volumes are termed variable; however, only one volume may be used at a time. ▪ The available range of pipette volumes is 1 μL to 5000 mL. ▪ A pipette with a capability of less than 1 mL is considered a micropipette, and a pipette that dispenses greater than 1 mL is called an automatic macropipette. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Multichannel pipettes are able to attach multiple pipette tips to a single handle and can then be used to dispense a fixed volume of fluid to multiple wells, such as to a multiwell microtiter plate. ▪ In addition to classification by volume delivery amounts, automatic pipettes can also be categorized according to their mechanism: air displacement, positive displacement, and dispenser pipettes. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ An air-displacement pipette relies on a piston for creating suction to draw the sample into a disposable tip that must be changed after each use. The piston does not come in contact with the liquid. ▪ A positive-displacement pipette operates by moving the piston in the pipette tip or barrel, much like a hypodermic syringe. It does not require a different tip for each use. Because of carryover concerns, rinsing and blotting between samples may be required. ▪ Dispensers and dilutor/dispensers are automatic pipettes that obtain the liquid from a common reservoir and dispense it repeatedly. The dispensing pipettes may be bottle-top, motorized, handheld, or attached to a dilutor. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ The dilutor often combines sampling and dispensing functions. Many automated pipettes use a wash between samples to eliminate carryover problems. ▪ However, to minimize carryover contamination with manual or semiautomatic pipettes, careful wiping of the tip may remove any liquid that adhered to the outside of the tip before dispensing any liquid. Care should be taken to ensure that the orifice of the pipette tip is not blotted, drawing sample from the tip. ▪ Another precaution in using manually operated semiautomatic pipettes is to move the plunger in a continuous and steady manner. Pipettes should be operated according to the manufacturer’s directions. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Laboratory Glassware ▪ Disposable, one-use pipette tips are designed for use with air- displacement pipettes. ▪ The laboratorian should ensure that the pipette tip is seated snugly onto the end of the pipette and free from any deformity. Plastic tips used on air-displacement pipettes can vary. ▪ Different brands can be used for one particular pipette, but they do not necessarily perform in an identical manner. ▪ Tips for positive-displacement pipettes are made of straight columns of glass or plastic. These tips must fit snugly to avoid carryover and can be used repeatedly without being changed after each use. As previously mentioned, these devices may need to be rinsed and dried between samples to minimize carryover. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Desiccators and Desiccants ▪ Many compounds combine with water molecules to form loose chemical crystals. ▪ The compound and the associated water are called a hydrate. When the water of crystallization is removed from the compound, it is said to be anhydrous. ▪ Substances that take up water on exposure to atmospheric conditions are called hygroscopic. ▪ Materials that are very hygroscopic can remove moisture from the air as well as from other materials. ▪ These materials make excellent drying substances and are sometimes used as desiccants (drying agents) to keep other chemicals from becoming hydrated. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Desiccators and Desiccants ▪ Closed and sealed containers that include desiccant material are referred to as desiccators and may be used to store more hygroscopic substances. ▪ Many sealed packets or shipping containers, often those that require refrigeration, include some type of small packet of desiccant material to prolong storage. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Balances ▪ A properly operating balance is essential in producing high-quality reagents and standards. However, because many laboratories discontinued in-house reagent preparation, balances may no longer be as widely used. ▪ Balances are classified according to their design, number of pans (single or double), and whether they are mechanical or electronic or classified by operating ranges. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Balances ▪ Analytic and electronic balances are currently the most popular in the clinical laboratory. ▪ Analytic balances are required for the preparation of any primary standard. ▪ It has a single pan enclosed by sliding transparent doors, which minimize environmental influences on pan movement, tared weighing vessel, and sample. ▪ An optical scale allows the operator to visualize the mass of the substance. The weight range for many analytic balances is from 0.01 mg to 160 g. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Balances ▪ Electronic balances are single-pan balances that use an electromagnetic force to counterbalance the weighed sample’s mass. ▪ Their measurements equal the accuracy and precision of any available mechanical balance, with the advantage of a fast response time (< 10 seconds). CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Centrifuges ▪ Centrifugation is a process in which centrifugal force is used to separate serum or plasma from the blood cells as the blood samples are being processed; to separate a supernatant from a precipitate during an analytic reaction; to separate two immiscible liquids, such as a lipid-laden sample; or to expel air. ▪ When samples are not properly centrifuged, small fibrin clots and cells can cause erroneous results during analysis. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Centrifuges ▪ The centrifuge separates the mixture based on mass and density of the component parts. ▪ It consists of a head or rotor, carriers, or shields that are attached to the vertical shaft of a motor or air compressor and enclosed in a metal covering. ▪ The centrifuge always has a lid, with new models having a locking lid for safety. Many models include a brake or a built-in tachometer, which indicates speed, and some centrifuges are refrigerated. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Centrifuges ▪ Centrifugal force depends on three variables: mass, speed, and radius. The speed is expressed in revolutions per minute (rpm), and the centrifugal force generated is expressed in terms of relative centrifugal force (RCF) or gravities (g). ▪ The speed of the centrifuge is related to the RCF by the following equation: ▪ where 1.118 × 10−5 is a constant, determined from the angular velocity, and r is the radius in centimeters, measured from the center of the centrifuge axis to the bottom of the test tube shield or bucket. CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Centrifuges ▪ Centrifuge maintenance includes daily cleaning of any spills or debris, such as blood or glass, and ensuring that the centrifuge is properly balanced and free from any excessive vibrations. Balancing the centrifuge load is critical. ▪ Many newer centrifuges will automatically decrease their speed if the load is not evenly distributed, but more often, the centrifuge will shake and vibrate or make more noise than expected. ▪ A centrifuge needs to be balanced by equalizing both the volume and weight distribution across the centrifuge head. ▪ Many laboratories will have “balance” tubes of routinely used volumes and tube sizes, which can be used to match those from patient samples. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Centrifuges ▪ The centrifuge cover should remain closed until the centrifuge has come to a complete stop to avoid any aerosol production. ▪ It is recommended that the timer, brushes (if present), and speed be periodically checked. ▪ The brushes, which are graphite bars attached to a retainer spring, create an electrical contact in the motor. ▪ The specific manufacturer’s service manual should be consulted for details on how to change brushes and on lubrication requirements. ▪ The speed of a centrifuge is easily checked using a tachometer or strobe light. CLINICAL CHEMISTRY CHAPTER 1 Laboratory Equipment o Centrifuges ▪ The hole located in the lid of many centrifuges is designed for speed verification using these devices but may also represent an aerosol biohazard if the hole is uncovered. Accreditation agencies require periodic verification of centrifuge speeds. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o The process of specimen collection, handling, and processing remains one of the primary areas of preanalytic errors. o Careful attention to each phase of the testing process is necessary to ensure proper subsequent testing and reporting of accurate and reliable results. o All accreditation agencies require laboratories to clearly define and delineate the procedures used for proper collection, transport, and processing of patient samples and the steps used to minimize and detect any errors, along with the documentation of the resolution of any errors. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Phlebotomy, or venipuncture, is the act of obtaining a blood sample from a vein using a needle attached to a collection device or a stoppered evacuated tube. ▪ The collection tubes are available in different volume sizes: from pediatric sizes (≈150 μL) to larger 10 mL tubes. ▪ The most frequent site for venipuncture is the medial antecubital vein of the arm. ▪ A tourniquet made of pliable nonlatex rubber flat band or tubing is wrapped around the arm, causing cessation of blood flow and dilation of the veins, making for easier detection. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ The gauge of the needle is inversely related to the size of the needle; the larger the number, the smaller the needle bore and length. Routine venipuncture uses a 23- or 21-gauge needle. ▪ A winged infusion set, sometimes referred to as a butterfly because of the appearance of the setup, may be used whenever the veins are fragile, small, or difficult to detect. ▪ The butterfly needle is attached to a piece of tubing, which is then attached to a hub or barrel. Because of potential for needlesticks and cost of the product, this practice may be discouraged. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ When selecting an appropriate vein, sites adjacent to IV (intravenous) therapy should be avoided. ▪ If both arms are involved in IV therapy and the IV cannot be discontinued for a short time, a site below the IV site may be considered. ▪ In such cases, the initial sample drawn (5 mL) should be discarded because it is most likely contaminated with IV fluid and only subsequent sample tubes should be used for analytic purposes. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ In addition to venipuncture, blood samples can be collected using a capillary puncture technique that involves using either the outer area of the bottom of the foot (a heel stick) for infants or the lateral side of the middle or ring finger for individuals 1 year and older (finger stick). ▪ A sharp lancet device is used to pierce the skin, and an appropriate capillary or microtainer tube is used for sample collection. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Analytic testing of blood involves the use of whole blood, serum, or plasma. ▪ Whole blood, as the name implies, contains the liquid portion of the blood, called plasma, and its cellular components (red blood cells, white blood cells, and platelets). ▪ The collection of whole blood requires the vacuum tube to contain an anticoagulant. ▪ Complete mixing of the blood immediately following venipuncture is necessary to ensure the anticoagulant adequately inhibits the specimen from clotting. As the tube of whole blood settles, the cells fall toward the bottom, leaving a clear yellow supernatant on top, which is the plasma. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ If a tube does not contain an anticoagulant, the blood forms a fibrin clot incorporating the cells; this clot consumes fibrinogen. ▪ The remaining liquid is called serum rather than plasma. ▪ Most testing in the clinical chemistry laboratory is performed on either plasma or serum. The major difference between plasma and serum is that serum does not contain fibrinogen and some potassium is released from platelets (i.e., potassium levels are slightly higher in serum than in plasma). ▪ It is important that serum samples be allowed to completely clot (≈30 minutes) in an upright position at room temperature before being centrifuged. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Plasma samples also require centrifugation but do not need time to clot, decreasing the turnaround time for testing and reporting results. ▪ Centrifugation of the sample accelerates the process of separating the liquid portion and cellular portion. ▪ Specimens should be centrifuged according to recommendations by the tube manufacturer or test protocol, usually approximately 10 minutes at an RCF of 1000 to 2000 g, but should avoid the mechanical destruction of red blood cells that can result in hemoglobin release, which is called hemolysis. CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Arterial blood samples measure blood gases (partial pressures of oxygen and carbon dioxide) and pH. ▪ Syringes containing heparin anticoagulant are used instead of evacuated tubes because of the pressure in an arterial blood vessel. ▪ The radial artery is the primary arterial site, while there may be times when the brachial or femoral artery may be considered. Arterial punctures are more difficult to perform because of inherent arterial pressure, difficulty in stopping bleeding afterward, and the undesirable development of a hematoma, which cuts off the blood supply to the surrounding tissue. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Proper patient identification is the first step in sample collection. The importance of using the proper collection tube, avoiding prolonged tourniquet application, drawing tubes in the proper order, and proper labeling of tubes cannot be stressed strongly enough. Prolonged tourniquet application causes a stasis of blood flow and an increase in hemoconcentration and anything bound to proteins or the cells. ▪ Having patients open and close their hand during phlebotomy is of little value and may cause an increase in potassium or lactic acid and, therefore, should be avoided. ▪ IV contamination should be considered if a large increase occurs in the substances being infused, such as glucose, potassium, sodium, and chloride, with a decrease of other analytes such as urea and creatinine. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Isopropyl alcohol wipes, for example, are used for cleaning and disinfecting the collection site; however, isopropyl alcohol is not recommended for disinfecting the site when drawing blood alcohol levels (in such cases, chlorhexidine is used as the disinfectant). CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Blood is not the only sample analyzed in the clinical chemistry laboratory. Urine is the next most common fluid for determination. ▪ Most quantitative analyses of urine require a timed sample (usually 24 hours); a complete sample (all urine collected within the specified time) can be difficult because many timed samples are collected by the patient in an outpatient situation. ▪ Creatinine analysis is often used to assess the completeness of a 24-hour urine sample because creatinine output is relatively free from interference and is stable, with little change in output within individuals. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ The average adult excretes 1 to 2 g of creatinine per 24 hours. Urine volume differs widely among individuals; however, a 4-L container is adequate (average output is ≈2 L). It should be noted that this analysis differs from the creatinine clearance test used to assess glomerular filtration rate, which compares urine creatinine output with that in the serum or plasma in a specified time interval and urine volume (often correcting for the surface area). CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Other body fluids analyzed by the clinical chemistry laboratory include cerebrospinal fluid (CSF), paracentesis fluids (pleural, pericardial, and peritoneal), and amniotic fluids. The color and characteristics of the fluid before centrifugation should be noted for these samples. Before centrifugation, a laboratorian should also verify that the sample is designated for clinical chemistry analysis only because a single fluid sample may be shared among several departments (i.e., hematology or microbiology), and centrifugation could invalidate other laboratory testing in those areas. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ CSF is an ultrafiltrate of the plasma and is approximately two- thirds of the plasma glucose value. ▪ For glucose and total protein analysis, it is recommended that a blood sample be analyzed concurrently with the analysis of those analytes in the CSF. ▪ This will assist in determining the clinical utility of the values obtained on the CSF sample. ▪ This is also true for lactate dehydrogenase and protein assays requested on paracentesis fluids. All fluid samples should be handled immediately, without delay between sample procurement, transport, and analysis. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Types of Samples ▪ Amniotic fluid may be used to assess fetal lung maturity, congenital diseases, hemolytic diseases, genetic defects, and gestational age. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ When samples arrive in the laboratory, they are first processed. In the clinical chemistry laboratory, this means correctly matching the blood collection tube(s) with the appropriate test requisition and patient identification labels. This is a particularly sensitive area of preanalytic error. ▪ Bar code labels (either as 1D linear barcodes, or 2D QR barcodes) or radiofrequency ID chip labeling on primary sample tubes are vital in detecting errors and to minimizing clerical errors. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ The laboratorian must also ascertain if the sample is acceptable for further processing. The criteria used depends on the test involved but usually include volume considerations (i.e., is there sufficient volume for testing needs?), use of proper anticoagulants or preservatives (i.e., was it collected in the correct evacuated tube?), whether timing is clearly indicated and appropriate for timed testing, and whether the specimen is intact and has been properly transported (e.g., on ice, within a reasonable period, protected from light). CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ Unless a whole blood analysis is being performed, the sample is then centrifuged as previously described and the serum or plasma should be separated from the cells if not analyzed immediately. ▪ Today, the use of serum separator tubes and plasma separator tubes is common practice. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ Once the sample is processed, the laboratorian should note the presence of any serum or plasma characteristics such as hemolysis and icterus (increased bilirubin pigment) or the presence of turbidity often associated with lipemia (increased lipids). ▪ Many analytes are stable at room temperature between 24 to 72 hours. However, if testing is not to be performed within 8 hours, it is recommended that serum and/or plasma be refrigerated between 2°C and 8°C. ▪ It is important to avoid exposing samples that are light sensitive, such as bilirubin, to artificial or ultraviolet light for extended periods of time. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ Separated serum and/or plasma may be frozen at −20°C and stored for longer periods without deleterious effects on the results. Repeated cycles of freezing and thawing, like those that occur in so-called frost-free freezers, should be avoided. ▪ Hemolysis can be visually observed in a centrifuged patient sample as a red color due to the release of hemoglobin. ▪ There are patient conditions where this may occur in vitro, such as hemolytic anemia, but hemolysis can also be present due to preanalytic collection variables such as inappropriate needle gauge, venipuncture site selection (small veins), and venous trauma or difficulty in specimen collection. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ Along with the release of hemoglobin, other intracellular components may be released, such as potassium, phosphate, and lactate dehydrogenase, which may impact patient values for these analytes. For analyzers utilizing spectrophotometric or enzymatic detection methods, hemolysis may also cause errors during assay. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ Lipemia results when the lipid levels of the patient are elevated and, in turn, is visualized as a creamy or milky appearance to the serum or plasma upon centrifugation. Lipemia can cause a volume displacement for some analytes, such as electrolytes, as well as interference in light-scattering methodologies due to the turbidity present. ▪ Icterus is a deep yellow or golden appearance of the serum or plasma due to increased bilirubin levels, and may cause spectral interference on certain analyzers in the chemistry lab. ▪ To help determine if interference has occurred, many analyzers are capable of detecting, then estimating, the interferent and the effect on sample values. CLINICAL CHEMISTRY CHAPTER 1 Specimen Collection and Handling o Sample Processing ▪ Lipemia results when the lipid levels of the patient are elevated and, in turn, is visualized as a creamy or milky appearance to the serum or plasma upon centrifugation. Lipemia can cause a volume displacement for some analytes, such as electrolytes, as well as interference in light-scattering methodologies due to the turbidity present. ▪ Icterus is a deep yellow or golden appearance of the serum or plasma due to increased bilirubin levels, and may cause spectral interference on certain analyzers in the chemistry lab. ▪ To help determine if interference has occurred, many analyzers are capable of detecting, then estimating, the interferent and the effect on sample values. CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1 CLINICAL CHEMISTRY CHAPTER 1