Laboratory Basics - Chapter 1 PDF

2 CHAPTER 1 LAboRAToRy bAsiCs Key Terms Atomic mass unit (amu) International unit (IU) Relative centrifugal Revolution...

2 CHAPTER 1 LAboRAToRy bAsiCs Key Terms Atomic mass unit (amu) International unit (IU) Relative centrifugal Revolution per minute Avogadros number Molarity (M) force (RCF) (RPM) Density Normality (N) Resistivity Thermocouple Gram molecular weight (GMW) Pascal (Pa) Reverse osmosis (RO) Torr A CASE IN POINT The laboratory supervisor discovered that the quality-control Issues and Questions to Consider (QC) results for serum creatinine in both levels of serum-based quality-control material had moved down-ward 1. Identify several causes for the downward move-ment below the mean on a consistent basis. This move-ment of quality-control results. of QC results either above or below the mean on a 2. Indicate the type of pipette(s) that is suitable for consistent basis is known as a QC shift. The quality-control reconstituting quality-control material. material is lyophilized (freeze-dried) and requires reconsti-tution 3. Are the procedures and techniques required to use with 5.0 mL of the diluent provided by the manu-facturer. pipettes the same for every pipette? Aninvestigation into the cause of this downward movement in quality-control results wasinitiated. Whats Ahead 1. Discussion of equipment commonly used in clinical chemistry 3. Identification of laboratory mathematics associated with several laboratories. functions performed in the laboratory. 2. Identification of reliable resources for operating, calibrating, and 4. Examples of laboratory mathematic problems with solutions. documenting use of basic equipment in the laboratory. INTRODUCTION to flush and clean internal components of analyzers, Laboratory testing requires technical skills and the ability to follow to serve as a medium in heating bath to incubate cuvettes, procedures. The clinical laboratory technologist (CLS) needs to be and familiar with fundamental practices and the equipment used in labora-tory to wash and rinse laboratory glassware. testing so that he or she may successfully complete a testing pro-cedure. For manyof these uses,the source of water mustbe of the These fundamental practices include selecting the proper types highest purity, whereasthe waterrequired for rinsing generallabo-ratory of pipettes, pipette use, centrifugation, laboratory calculations, tem-perature glassware may beless pure. monitoring, and others that will be presented in this chapter. Another significant aspect associated with equipment familiar-ization is calibrate and calibration. These terms are used throughout Clinical Laboratory Reagent Water this chapter and others. The term calibrate (a verb) refers to tasks Clinical and Laboratory Standards Institute (CLSI), formerly the of assessing, setting, or correcting a device (e.g., thermometer, cen-trifuge, National Committee for Clinical Laboratory Standards (NCCLS), balance, or spectrometer) usually by comparing or adjust-ing has developed specifications for laboratory water quality. The it to match or conform to a reliable, known, and unvarying criteria for clinical laboratory reagent water (CLRW) are outlined in measure. For specific examples, see the sections on thermometry, CLSI, GP40-A4-AMD guidelines1 and can be found in Bermes centrifuges, pipettes, and balances. Calibration refers to the actual et al.2 Whenselecting a water-purification system, the purchaser process used to calibrate a device. must review the criteria established by CLSI so that all of the appropriate filters and components necessary to produce CLRW WATER are included. Also, the source of the feedwater or tap water that Waterhas numerous usesin the clinical laboratory, including the will be purified should be determined. The feedwater may contain following: unique contaminants or may have a high mineral content (hard-ness), which will often require the inclusion of additional compo-nents in the preparation of reagents, in the water-processing system designed to remove these as a diluent for controls and calibrators, substances  CHAPTER 1 LAboRAToRy bAsiCs 3 Purification BOx 1-1 Criteria for Clinical Laboratory Many laboratories produce or purify their own water. There are Reagent Water (CLRW). several water-purification systems available to produce CLRW. Most water-filtration systems use a prefilter to begin the process. Resistivity, MO cm, at 25C 710 This prefilter has feedwater running through it to trap any particu-lates Microbiological impurities, Colony forming units per mL before the water is sent on to the next process, which is either (Cfu/mL) maximum 610 distillation or reverse osmosis. Distillation is the process by which Organics, total Organic Carbon (TOC), ng/g (parts per aliquid is vaporized and condensed to purify or concentrate a sub-stance billion, ppb) 6500 or separate volatile substances from less-volatile substances. Silicates, mg SiO2/L 0.05 Water that is only distilled does not meet the specific resistivity requirements for CLRW. Resistivity is the electrical resistance in Particulates, water passed through a 0.22 mm filter ohms measured between opposite faces of a 1.00-cm cube of an aqueous solution at a specified temperature for CLRW. Additional filters (e.g., ion exchange, carbon material, and particulates) are of these parametersshould be checkedon a periodic basis.Box 1-1 required to produce CLRW water and can be arranged so that the lists several examples of water purity factors that are commonly distalate passes through them. Reverse osmosis (RO) is a pro-cess monitored and acceptable cutoff values for CLRW.1,2 by which water is forced through a semipermeable membrane Most water-filtration systems will havea resistivity meter that acts as a molecular filter. The RO filter removes 9599% of placed or plumbed in-line, that is, so the filtered water can pass organic compounds, bacteria, and other particulate matter and directly through it and out to the next filter or be dispensed. Resis-tivity approximately 95% of allionized and dissolved minerals, but not measurementsare usedto assessthe ionic content of puri-fied as many gaseous impurities. RO alone does not result in the pro-duction water. Therefore, an inverse relationship exists between the of CLRW, butas with distillationif additional filters ion concentrations in water and the resistivity value: The higher are added (e.g., ion exchange, carbon, and particulates) to the sys-tem, the ion concentrationin water,the lower the resistivity value will CLRW water may be produced. be. CLSI requires that CLRW water have a resistivity greater than Ion-exchange filters remove ions to reduce the mineral con-tent 10M#cm. of deionized water. Deionization is accomplished by passing Monitoring bacterial contamination can be accomplished water through insoluble resin polymers that contain either anion-or quite easily. The water should be allowed to run for at least one cation-exchange resins. These resins exchange hydrogen ions minute to flush the system. Next, an aliquot of wateris obtained (H+) and hydroxyl (OH-) ions for ionized impurities present in and plated onto an appropriategrowth medium. Afteran appropri-ate the water. Another type of material used is a mixed bed resin that incubation time, the number of colony-forming units on the contains both anion-and cation-exchange materials. Ion-exchange agar plateis determined. Gram-negative rods are the most com-monly below) exceeding filters 1to10Mohm are capable of producing cm(also M#cm). AM#cm water that has a resistivity is (see found organismsin waterafter the purification process. described as mega (equivalent to 1,000,000 or one million) ohm (an Water Use electrical unit of resistance) centimeter. Carbon filters containing Most water-purification systems are designed for easy access to activated charcoal can be added to the water-purification system end-product CLRW, so it is advisable to use only CLRW water to help remove several types of organic compounds that may be for mostlaboratory procedures. Whenspecialchemistry testing in the water. is required (e.g., assay of heavy metals,and analyte testing using A particulate or bacteria filter can be added at the end of the high-performance liquid chromatography (HPLC)) the labora-tory system. This filter with a mean pore size of about 0.22 mm will should usespecialreagent water(SRW) and CLRWfor those serve to trap any remaining particulates including bacteria aslarge procedures. as or larger than the pore size. The criteria usedto specify CLRW should beincluded in SRW specifications. Thesespecial applications mayrequire differ-ent Monitoring Water Purity limits, so additional parameters maybe added if necessary. For example,the water mayhaveto be passedthrough morethan one Because water is such an integral part of laboratory analysis, its carbon filter or the pore size of the bacteria filter mayhave to be purity must be monitored on a consistent basis. The frequency of smaller. water monitoring and testing depends on many factors, includ-ing the composition of the feedwater, availability of staff to per-form the water testing, and the amount of water the laboratory uses during a given period of time. Many laboratories do not CHEMICALS have the resources to conduct several procedures for complete The chemicals used to prepare reagents for testing exist in vary-ing assessment of water quality but, at a minimum, should monitor degrees of purity. Proper selection of chemicals is important resistivity and bacterial content of the water on a regular basis. so that the desired results may be attained. Chemicals acquired In addition, pH, silica content, and organic contaminants may be for reagent preparation are characterized by a grading system. The determined. Depending on the laboratory resources, some or all grading of any chemical is greatly influenced byits purity. The typ  4 CHAPTER 1 LAboRAToRy bAsiCs and quantity of impurities are usually stated on the label affixed is six times stronger than borosilicate glass.For example, Corex to the chemical container. Less-pure grades of chemicals include pipettes have a typical strength of 30,000 pounds per square inch practical grade, technical grade, and commercial grade; all of them (psi), compared with 2000 to 5000 psi for borosilicate pipettes and are unsuitable for use in many quantitative assays. will outlast conventional glasswaretenfold. Corex also resists Most qualitative and quantitative procedures performed in clouding and scratching better than other types of glassware. the clinical laboratory require the use of chemicals that meet the (NOTE: Many clinical laboratories may continue to have specifications of the American Chemical Society. These chemicals Corex brandglasswarein their laboratories.) are classified as either analytical grade or reagent grade. Examples Lowactinicglasswareis a glass of high thermal resistance withan of other designations of chemicals that meet high standards of amber or red color added as an integral part of the glass. The density purity include spectrograde, nanograde, and HPLC grade. These of the red color is adjusted to permit adequate visibility of the con-tents are often referred to as ultrapurechemicals. yet give maximum protection to light-sensitive materials such Pharmaceutical chemicals are produced to meetthe specifica-tions as bilirubin. Low actinic glass is commonly used in containers for definedin The UnitedStatesPharmacopeia, The NationalFormu-lary,control material, calibrators, and reagents. A comparison between and TheFood ChemicalIndex. The specifications define impurity clear glass and low actinic glass is shown in Figure 1-1. tolerances that are not injurious to health. The International Union for Pure and Applied Chemistry Types of Plasticware (IUPAC) has developed standards and purity levels for certain Several types of plastics are used in clinical laboratories. Examples chemicals. These include atomic weight standard (grade A), ulti-mate include polypropylene, polyethylene, polycarbonate, and polysty-rene. standard (grade B), primary standard (grade C), working stan-dard Plastics are used for pipette tips, beakers, flasks, cylinders, (grade D), and secondary substances (grade E). and cuvettes. A very good source of highly purified chemicals, especially Plastic pipette tips are made primarily of polypropylene. This reference materials, is the National Institute of Standard and Test-ing type of plastic may be flexible or rigid, is chemically resistant, and (NIST, Gaithersburg, MD). NIST defines its chemical and can be autoclaved. These pipette tips are translucent and come in a physical properties for each compound and provides a certificate variety of sizes. Polypropylene is also used in several tube designs, documenting their measurements. NIST also provides standard including specimen tubes and test tubes. Specially formulated reference materials (SRMs) in solid, liquid, or gaseous form. The polypropylene is used for cryogenic procedures and can withstand solids may be crystalline, powder, or lyophilized. temperatures aslow as -190C.3 CLSI and the College of American Pathologists (CAP) are two Polyethylene is widely used in plasticware for test tubes, professional organizations that can provide laboratory staff with bottles, graduated tubes, stoppers, disposable transfer pipettes, guidelines for proper chemical selection and reagent preparation. volumetric pipettes, and test-tube racks. Polyethylene may bind or There may be membership requirements for these organizations in absorb proteins, dyes, stains, and picric acid, so care must be taken order to use their guidelines. Their respective websites will provide before selecting polyethylene. Polycarbonate is used in tubes for the user with pertinent information to access their resources. centrifugation, graduated cylinders, and flasks. The usable tem-perature rangeis broad: -100 to +160C. It is a verystrong plastic LABORATORY GLASSWARE but is not suitable for use with strong acids, bases, and oxidizing AND PLASTICWARE agents. Polycarbonate may be autoclaved but with limitations (refer to the instructions provided by the manufacturer).3 Types of Glassware Borosilicate glass is commonly used in clinical laboratories and can be found in beakers, flasks, and test tubes. This glass is characterized by a high degree of thermal resistance; it has low alkali content and is free of heavy metals. Commercial brands include Pyrex (intro-duced by Corning, Corning, NY) and Kimax (Kimble-Chase, Vineland, NJ). The caustic properties of concentrated alkaline solutions in borosilicate glass will etch or dissolve the glass and destroy the calibration. Borosilicate glassware with heavy wallssuch as bottles, jars, and even large beakersshould not be heated with a direct flame or hot plate. Glass should not be heated above its strain pointfor example, the temperature for Pyrex is 515Cbecause rapid cooling strains the glass, which will crack easily when heated again. In the case of volumetric glassware, heat-ing can destroy the calibration. Corex* (Corning, NY) brand glasswareis a special alumina-silicate glassstrengthened chemically rather than thermally. Corex AB FIGURE 1-1 Types of glass bottle containers. A. * Corning Glass no longer manufactures Corex glassware. Transparent glass and B.low actinic glass  CHAPTER 1 LAboRAToRy bAsiCs 5 Polystyrene is a rigid, clear type of plastic that should not be Transfer Pipette Transfer pipettes include both volumetric and autoclaved. It is used in an assortment of tubes, including capped Ostwald-Folin pipettes. These pipettes consist of a cylindrical bulb graduated tubes and test tubes. Polystyrene tubes will crack and at both ends to narrower glass tubing. A calibration markis etched splinter when crushed; thus, care must be taken when handling around the upper suction tube, and the lower deliver tube is drawn damaged tubes. This type of plastic is not resistant to most hydro-carbons,out to a gradual taper. ketones, and alcohols. A volumetric transfer pipette (see Figure 1-2C ) is calibrated Teflon is a brand name for polytetrafluoroethylene (PTFE) to deliver accurately a fixed volume of an aqueous solution. The and is widely used for manufacturing stirring bars, tubing, cryo-genic reliability of the calibration of the volumetric pipette decreases vials, and bottle-cap liners. Teflon is almost chemically inert with a decrease in size; thus, micropipettes, discussed below and is suitable for use at temperatures ranging from -270 to have been developed and replaced most glass and plastic transfer +255C. Thistype of material is resistantto a widerange of chem-ical pipettes. They are designed to dispense smaller volumes of liquids classes, including acids, bases, alcohols, and hydrocarbons. accurately and precisely. Ostwald-Folin pipettes are similar to volumetric pipettes but have their bulb closer to the delivery tip and are useful for the Volumetric Laboratoryware accurate measurement of viscous fluids such as blood or serum. Pipettes In contrast to the volumetric pipette, an Ostwald-Folin pipette Many types of pipettes are available for use in a clinical laboratory, has an etched ring near the mouthpiece, indicating that it is a and each is intended to serve a specific function. Pipettes are used blowout pipette. A pipette bulb should be used to aid in the expul-sion to reconstitute lyophilized controls and calibrators, prepare serum of the blood sample after the blood has drained to the last and plasma dilutions, and aliquot specimens. Thus, a high degree drop in the delivery tip. When filled with opaque fluids, such as of accuracy and precision is required. Volumentric pipettes fall into blood, the top of the meniscus must be read. The drainage of two general categories: transfer (volumetric) and measuring. Three the blood should be controlled so that no residual film is left on subclassifications include to contain(TC), to deliver(TD), andto deliver/ the walls of the pipette. blowout (TD/blowout). (See below.) Apipetting aid,(e.g.,bulb) must be used when pipetting solutions using glass or plastic pipettes. Measuring Pipettes The second principal type of pipette is the graduated or measuring pipette. These pipettes consist of Class A Designation Class A glassware, including pipettes, is a piece of glass tubing that is drawn out to a tip and graduated manufactured and calibrated to deliver the most accurate volume uniformly along its length. Examples include the Mohr pipette of liquid. Class A specifications are defined by NIST. CAP speci-fies (Figure 1-2D ), which is calibrated between two marks on the that volumetric pipettes must be of certified accuracy (class stem, and the serological pipette (Figure 1-2B ), which must be A) by the manufacturer or the volumes of the pipettes must be blown out to deliver the entire volume of the pipette and has an verified by calibration techniquesfor example, gravimetric or etched ring near the bulb. Typically two etched lines are present, photometric. The letter A appears on all pipettes that conform which signify that it is a blowout pipette. Mohr pipettes require to the standards of class A glassware. Volumetric glassware des-ignated a steady, controlled delivery of the solution between the calibra-tion as class A has been manufactured to class Atolerances as marks. Serological pipettes have a larger orifice than do the established by American Standards and Testing Materials (ASTM) Mohr pipettes and therefore drain faster. In clinical laboratories, (West Conshohocken, PA) for volumetric ware. measuring pipettes are typically used for the measurement of reagents and are not generally considered sufficiently accurate Types of Pipettes for measuring samples and calibrators Two commonly used pipettes in clinical laboratories are transfer and measuring. Atransfer pipette is designed to transfer a known volume of liquid. Measuring and serological pipettes are scored in units such that any volume up to a maximum capacity is delivered. The accuracy of these types of pipette is a significant factor when it is necessary to select an appropriate pipette to use. The accuracy tolerances for selected pipettes shown in Example 1-1 below high-light differences between classes and types of pipettes.4,5 ExampLE 1-1 The accuracy tolerance for a 1.0 mL class A volumetric transferpipetteis {0.006 mL;for a classB1.0 mLvolu-metric FIGURE 1-2 Several examples of glass pipettes. transferpipettes,it is {0.012 mL.Theaccuracy A. 0.2 mL TC; B. 1.0 mL TD serologic (blowout); tolerance for a 1.0 mL class B measuring and serological C. 2.0 mL TD volumetric; and D. 10.0 mL TD Mohr. pipette is 0.02 mL. Note the two frosted-or etched-glass rings on pipette B. 6 CHAPTER 1 LAboRAToRy bAsiCs ChECkpoInt! 1-1 BOx 1-2 Specific features of pipettes Identify which type of glass pipette would be the best shown in Figure 1-3. to use to reconstitute lyophilized, serum-based, quality-control material. A Finnpipette Air displacement, variable volume selection, one-stroke plunger action, tip ejector B PipetmanTM Air displacement, fixed and vari-able Micropipettes Two examples of commonly used micropipettes volume selection, one-stroke are air-displacement and positive-displacement micropipettes. plunger action, color-code system These pipettes are capable of delivering liquid volumes from for easy identification of pipette 1 -1000 mL.Somemicropipettes aredesigned to deliverafixed volumes, tip ejector volume, while otherscan deliver variable amounts ofliquid. C Eppendorf Air displacement, fixed and vari-able An air-displacement micropipette uses a piston device to facil-itate volume selection, two-stroke aspiration and ejection of liquids. A disposable, one-time-use plunger action, tip ejector polypropylenetip is attachedto the pipette barrel. The pipettetip is D MLA Air displacement, fixed and vari-able placedinto the liquid to be aspirated and drawn into and dispensed volume selection, one-or from this tip. A positive-displacement micropipette uses a capillary two-stroke plunger action is avail-able, tip madeof glass or plasticto transfer liquids. A Teflon-coated color coded by volume tipped plunger fits tightly inside the capillary. Liquid solutions are drawn up the capillary and pushed out with a squeegee effect, thus E SMI Positive displacement, fixed and limiting the amount of carryover. Thesecapillarytips arereusable variable volume selection, one-stroke and suited for rinsing out solutions. Some procedures require a plunger action washing or flushing step between samples. Severalexamplesof micropipettescommonly usedin clinical laboratories are shown in Figure 1-3. Each of these micropi-pettesbe maintained.Thesepipettesalso havesealsto preventair from possesses operational features in its design; for example, air leaking into the pipette when the piston is moved. These seals displacementor positive displacement,fixed or variable volume require periodic lubrication to maintain their integrity. Positive-displacement selection, tip ejectors, and one-or two-stroke plunger action. Spe-cific micropipettesneedto havetheir spring checkedand examples of these features for each pipette displayedin Figure the Teflon tip replaced periodically. A slide wireis usedto quickly 1-3 are presentedin Box 1-2. check the plunger setting. This check does not replace the sched-uled precision and accuracychecks. Pipette Calibration Monitoringthe performance of pipetting Several procedures are used bylaboratories to verify precision devices is required in mostlaboratories licensed bytheir respective and accuracy of micropipettes. Most of these procedures aretime-consuming, states. Micropipettesshould be verified for accuracyand precision especiallythe proceduresthat requirethe weighingof before they are putinto use and monitored during the course of the water.It maytake several hours to properly evaluate precision and year. The frequency of verification depends in part on how exten-sivelyaccuracy of all pipettes used in the clinical laboratory because they are usedand requirements by the licensing oraccredit-ing each pipette requires multiple weighingsand proper monitoring agency. Proper maintenance of air-displacement pipettesis very of environmental factors such astemperature, humidity, and atmo-spheric important. This type of pipette hasa fixed stroke length that must pressure. These verification procedures must be performed no matter whatadverseimpact it hason the laboratory to ensure proper performance of laboratory micropipettes. A CLSI has provided a gravimetric procedure that is acceptable for determining pipette accuracyand precision.6This gravimetric procedure is labor-intensive but does provide alow-cost meansof B complying with the regulations set forth bythe various accrediting agencies. More expensive procedures for calibrating micropipettes C include: D commercial photometric pipette-calibration products, calibration-service providers, and E Pipette Tracker (Labtronics Inc., Guelph, ON, Canada). VC-100 AcidBase Titration Pipet Verification System,(Streck, Inc., Omaha, NE). FIGURE 1-3 Several examples of micropipettes used in clinical laboratories. PCS Pipette Calibration System (Artel, Westbrook, ME)  CHAPTER 1 LAboRAToRy bAsiCs 7 One major concern when considering the cost attributed to an analytical balance. The mass of an object is a measure of the pipette-verification procedures is clinical laboratory technologist amount of material in it as evidenced by its inertia. Inertia is a time. The clinical laboratory technologist time required for the measure of resistance to change of motion. The unit of mass photometric procedures is often 5060% less than the inexpensive commonly usedis the gram. Weightis a function of massunder manual-weighing techniques. the influence of gravity and is equal to mass multiplied by grav-ity. Thus, massis not the same as weight, even though we usually Volumetric Flasks determine an objects mass by measuring its weight. For example, Volumetric flasks are a special type of glassware in the laboratory. a man standing on the moon would weigh less than he would on The flask has a round flat bottom and along, thin neck with a cali-bration Earth because of the lower gravity on the moon, but he would line etched near the top. The last few milliliters should be have the same mass. added using a transfer-type pipette so that the meniscus to the cali-bration mark is not missed. These flasks are often used to prepare Types of Balances standard solutions for quantitative procedures, so their accuracy Several different types of balances are available, depending on is critical. Volumetric flasks used to prepare standards and other what needs to be weighed. For example, to weigh a fecal fat speci-men, solutions require optimal accuracy and must meet class A specifi-cations an appropriate balance would be a top-loading precision bal-ance established by ASTM in E1212-02.7 These specifications capable of accurately weighing kilogram amounts of sample. are imprinted on the flasks. Volumetric flasks are used to contain If a standard solution needs to be prepared for a toxicology assay an exact volume whenthe flaskis filled to the mark. A Teflon or that requires microgram quantities, then a single-pan microbalance ground glass stopper should be used to seal the flask. Volumetric is appropriate. flasks should not be used to store reagents. Unequal-Arm Substitution Balances Erlenmeyer Flasks Unequal-arm substitution balances are typically a single-pan design Erlenmeyer flasks may be graduated or not. They are designed to and are commonly used in laboratories, though electronic balances hold different volumes rather than one exact amount. These flasks are replacing almost all of these types of balances. This single-pan, are typically used to prepare reagents, so flask size, thermal stability, mechanical, unequal-arm balance operates on the principal of and chemical inertness should be considered. Erlenmeyer flasks removing weights rather than adding them. A fixed-mass counter-weight are often described as conical flasks because they have a wide is used to balance the combined mass of the pan and the flat bottom that gradually constricts to a smaller short neck. Their removable weights across two arms of unequal weight. When a specifications are describedin ASTM E1404-04.8 sample is placed on the weighing pan, the operator turns a set of knobs, which moves the internal weights in 1-g or 10-g increments. Griffin Beakers This adjustment is performed one increment at a time. This is con-tinued Griffin beakers have a variety of uses including preparation of until the system returns to equilibrium, at which time the reagents and as containers for a variety of liquids for many difference sum of the weights removed is equal to the weight of the object. purposes. They can be glass or plastic and hold different volumes rather than one exact amount. Griffin beakers, like the Erlenmeyer Magnetic ForceRestoration Balance flask, should be selected with size, thermal stability, and chemical Another commonly used balance is the single-pan balance that inertness in mind. The physical characteristics of a Griffin beaker are relies on magneticforce restoration. Restoring forceis the force a flat bottom, straight sides, and an opening that is as wide as the flat required to put the balance back into equilibrium. The object to base.It also has a small spout in the lip for safe pouring of liquids. be weighed is placed on the pan, and this system goes out of equi-librium. Specifications for Griffin beakersare describedin ASTM 1272-02.9 The operator adjusts the internal weights and restores partial equilibrium. A null-detector optics circuit senses when equi-librium Graduated Cylinders is near and provides a signal to the sensor motor to gener-ate Graduated cylinders are widely used in laboratories to measure a restoring current until equilibrium is reached. At this time, volumes of liquids. The measured volumes are not as accurate as the unknown massis equal to the mass of the weights removed volumetric glassware. The physical features of a graduated cylin-der plus the value of the restoring current. Standards for single-pan can be described as a long, cylindrical tube, typically standing mechanicalbalancescan befound in ASTM E319-85.11 upright on an octagonal or circular base. The cylinder has calibra-tion Top-Loading Balances marks along its length with different gradations depending on the design. The specifications for graduated cylinders are shown Single-pan, top-loading balances operate on the same principle in ASTM E 960-93.10 as single-pan analytical balances (i.e., weighing by substitution). Damping or releasing the pan is accomplished by magnetism rather than air release. These balances are especially suitable for quickly WEIGHING SUBSTANCES weighing larger masses(as much as 10,000 g)that do not require as Weighing substances is a fundamental process in preparing stan-dards much analytical accuracy, such asthe preparation of large volumes and reagents, performing gravimetric analysis, and calibrat-ing of reagent. Standards for top-loading balances can be found in volumetric laboratoryware. The process requires the use of ASTM EE898-88.1 8 CHAPTER 1 LAboRAToRy bAsiCs Electronic Balances There are several electronic balance designs. One design uses a strain-gaugeload cell. This small, thin device changeselectrical resistance when it is stretched or compressed. Typically, several strain gaugesare usedin a Wheatstonebridge arrangement and are glued onto the load cell in a protected location. Aload cell is usually in the shape of a beam or a plate. Whenthe beam or plate is displaced,it bendsa tiny amount; this tiny bendingis detected bythe strain gauges. The amount of bending might be only athou-sandth of aninch, but that is enough for a strain gaugeto measure. Another electronic balancedesign operateson the principle of electromagnetic force compensation. A coil placed between the poles of a cylindrical electromagnet is mechanically connected to a weighingpan. Whena substanceis placedonthe pan,it producesa FIGURE 1-4 ASTM standard weight set used to force that displaces the coil within the magneticfield. A regulator calibrate laboratory balances. generates a compensation current just sufficient to return the coil to its original position. The more weight placed on the pan,the should not be dragged across any surface, including the stain-less larger the deflecting force, and the stronger the current required to steel weighing pan. Usually the weights are sent in a specially correct the deflection of the coil. The measuring principle is based designed, covered box and should always be stored in that box. on a strict linear relationship betweencompensation current and ASTM provides calibration weights that range from a few milli-grams force produced bythe weight placed on the pan. to larger weights (e.g., 10 g). An example of a standard weight Several additional features may be available on some mod-els set used to calibrate laboratory balances is provided in Figure 1-4. of electronic balances.For example,some electronic balances Several factors may affect the performance of a laboratory include an electronic vibration damper. Any excess vibration can balance, including temperature, air drafts, floor vibrations, table be detected when a variation of the pointer or oscillation of the instability, and static electricity. Minimizing the effects of these numberin the last decimal placeof the digital displayis observed. factors on your weighing procedures can often be done quite eas-ily. Another feature available in some modelsis built-in taring. This For example, if there are air drafts in the room, a shroud or allows the weight of the weighing container to be automatically enclosure can be placed around the balance. A marble table can be subtractedfrom the total weight of the sample. Also, electronic used to reduce table vibrations or instability. balances can beinterfaced with computers to provide calculations such as weight averaging and statistical analysis of multiple weigh-ings. Balance Specifications Thefundamental design of electronic balancesallows for The laboratory staff should be familiar with the specifications faster weighing, whichis advantageous when performing multiple attributable to the respective balances. Examples of several impor-tant weighingsfor example,during a pipette precision procedure. balance specifications that an operator should be knowledge-able about include: Calibration of Balances capacity, the maximum load one can weigh; Laboratory balances require calibration at regular intervals. There is no fixed calibration interval for scientific applications, according accuracy,the closeness of the agreement between the measured to NIST. Calibrationintervals should coincide withthe require-ments result and the true value; of the laboratorys licensing and accrediting organizations. linearity, the ability of a balance to follow the linear relationship The mass-standard and test-weight-accuracy classes for betweenload andthe displayedvalue; weightsusedin calibrating balanceshavebeenupdatedand replace readability, the smallest increment of weight that can be read on the older requirements specified by National Bureau of Standards the display; and classes S and S1 weights. The new mass-standardsand test-weight-accuracy repeatability, the ability of a balance to produce the same result for classesappropriatefor laboratory balances include ASTM repeated weighing of the same load under the identical measure-ment classes 1 and 2. Referto ASTM E617-97 for specific information conditions. regarding range, readability, and best uncertainty applicable to these classes.13 Laboratory-accrediting agencies require that the accuracy of NIST class 1 weights (extra-fine accuracy) are available up to balances be verified at various time intervals. Consult your accred-iting 250 mgand maybe used for high-precision balancessuch assingle-pan agency for specific information regarding your equipment. and electronic balancesthat are preciseto four decimalplaces. The range of weight for class 2 balances may bein excess of 1000 grams (g). Meticulous care must be used when handling class 1 or CENTRIFUGES 2 weights.The operator mustavoid direct contact withthe weights Centrifuges serve an important role in preparing specimens for by using clean gloves or special lifting tools (for example, forceps). analysis. Improper centrifugation of specimens often leads to Hand contact with the weights can cause corrosion. The weights erroneous data. Assays, especially immunoassays, have very lo  CHAPTER 1 LAboRAToRy bAsiCs 9 Drive Shaft Rotor Assembly Converting revolutions per minute to RCF can also be derived from a nomogramthat is usuallyincluded in the manufacturers man-ual or found in many clinical chemistry textbooks. A nomogram is defined as a representation by graphs, diagrams, or charts of the relationship between numerical variables. Most centrifuges used in clinical laboratories operate by set-ting Drive Motor the speed in RPMs and then pushing the start button. Occa-sionally a laboratory procedure may state that the operator must FIGURE 1-5 Schematic showing the three major centrifuge the samples at a specific RCF. Therefore, if a proce-dure components of a typical laboratory centrifuge. requires the samples to be centrifuged at 1000 * g,simply use Equation 1-2 shown below or consult a clinical chemistry reference detectable levels for analytes in biological fluids and are prone to or textbook for a nomogram to makethe conversion. error caused by the presence of small fibrin clots and cells. The main components of a centrifuge are the motor, drive shaft, and rotor drive assembly, motor is as illustrated used to in provide Figure 1-5. the speeds required An elec-tromagnetic RPM = Ar* 1.118 RCF * 1000 (Eq. 1-2) to separate particulates from samples. These motors use carbon brushes to facilitate creation of electromagnetic fields that ulti-mately makethe drive shaft turn; this in turn spins the rotor assem-bly. Types of Centrifuges The buckets hold the tubes containing the samples. Several types of centrifuges are available to process specimens, separatelow-density particlesfor analysis,and clearspecimens of Relative Centrifugal Force and potential interfering compounds (lipids, for example). Examples Revolutions per Minute of the types of centrifuges include: Relative centrifugal force (RCF) is defined as a method of com-paring swinging-bucket rotor (also swing-out rotor with buckets), the force generated by various centrifuges on the basis of fixed-angle rotor, their speeds of rotation and distances from the center of rotation. Revolutions per minute (RPM) is a unit for expressing the num-ber air-driven ultracentrifuge, of complete rotations of a rotor occurring per minute. It is a ultracentrifuge, and measure of speed. refrigerated. Relative centrifugal force is expressed as some number times gravity (g). RCF is calculated using Equation 1-1. The swinging-bucket centrifuge shown in Figure 1-6A is routinely used to separate cells from serum or plasma. Both plain red-top tubes without serum-separator gel and serum-separator RCF = (1.118 * 10-5) (r) (rpm)2 (Eq. 1-1) tubes can be centrifuged. The required relative centrifuge force is 1000-1200 * g, and centrifuge times are between 5 and 10 minutes. The swinging-bucket design allows the tubes to assume where a horizontal position when the centrifuge is at maximum g force. 1.118 * 10-5 = empiricalfactor; During centrifugation, particulates (e.g., cells) constantly move along the tube while it is in the horizontal position. This move-ment r = radiusin centimetersfrom the center of rotation to the of particles spreads the sediment uniformly against the bot-tom bottom of the tube in the rotor cavity or bucket during of the tube. When the centrifuge rotor comes to a complete centrifugation; and stop, the surface of the sediment is flat with a column of serum rpm2 = total number of revolutions per minutesquared. or plasma above it. Fixed-angle rotors allow tubes to be centrifuged at angles ranging from 25 to 52, depending on the design. This rotor is ExampLE 1-2 shown in the top view in Figure 1-6B. The sample holder is placed in one of the six positions in the rotor. During centrifu-gation, Calculate the RCF of a centrifuge whose r is 10 cm from particles move along the side of the tube to form sedi-ment the center of rotation when the centrifuge is operated at that packs against the side and bottom of the tube. The a speed of 3000 rpm. surface of the sediment is parallel to the centrifuge shaft. Fixed-angle SoLutIon rotors are aerodynamically designed to yield much faster rotational speeds or greater g forces than swinging-bucket rotors. RCF = (1.118 * 10-5)(r)(rpm)2 A microcentrifuge used to prepare pellets of DNA and RNA RCF = (1.118 * 10-5 * 10 cm * (3,000 rpm)2 is equipped with a 25 or 45 fixed-angle rotor that can achieve RCF = 1006 * g RCFsof 18,000* g(14,000 RPMs).Anothertype of fixed-angle centrifuge that is widely used in the laboratory allows for a quic 10 CHAPTER 1 LAboRAToRy bAsiCs 2-minute spin at nearly 4400 * g(8,500 RPM)for 7.0-mL blood tubes. This type of centrifuge is being usedfor preparation of stat samples. The term statis from the Latin word statim, which means instantly/immediately. It is a directive to medical personnel during an emergency situation. In the clinical laboratory, it is a descriptor used to identify a sample that should be processed and measured in an urgent or rushed manner. The purpose of a 2-minute spin procedure is to reduce turnaround time for certain critical labora-tory tests. Most centrifuges use an electromagnetic motor to rotate the rotors. One exception to this design uses air to spin the rotor. This A centrifuge functions by directing compressed air onto grooves that are etched into the outer surface of the fixed-angle rotor. The rotor begins to move asthe air blows across the grooves, which are shown in the bottom and side views of Figure 1-6B. The maximum RCFis about 178,000 * g. Thistype of centrifugeis often usedto clear or remove lipid particles from lipemic specimens. Ultracentrifuges are muchlarger than regular laboratory cen-trifuges. They are often floor-model types and generate very high centrifugal forcesfor example, 800,000 * g(100,000 RPM). These high-speed centrifuges are used to fractionate lipoproteins, perform drug-binding assays, and prepare tissue for hormone-receptor assays. A refrigerated centrifuge is used routinely in laboratories for separations requiring colder temperatures. Temperature ranges from -15C to + 25C are achievable withthis type of centrifuge. Specimens for lactic-acid and plasma-ammonia determinations require the use of refrigerated centrifuges. Refrigerated centrifuges come equipped with either swinging buckets or fixed-angle rotors. Operation, Maintenance, and Calibration Proper operation of a laboratory centrifuge is an important func-tion in the preanalytical stage of testing. It must be done correctly and safely according to the manufacturers instructions. Once the specimens are loaded into the containers or buckets, they must adjusted so that the weight distribution is balanced. If the test tubes are not balanced, the centrifuge may not start or, if it does start, some tubes may break during centrifugation. Also, if the cen-trifuge has covers for the buckets, they should be used. Finally, all tubes and containers should be covered so that their contents do not leave the tubes and contaminate the inside of the centrifuge. Covering the patient specimens tubes also reduces the amount of fomites or particulates that may come from the samples. Routine maintenance of centrifuges includes cleaning the interior and exterior surfaces, the rotor, and the buckets with an appropriate disinfectant such as a 10% bleach solution. Any debris inside the centrifuge (for example, broken glass and stoppers) should be carefully removed. Centrifuge timers and speeds should be checked periodically for proper function. The timers can be checked against an accurate timepiece. Centrifugation speeds can be calibrated using a strobe B tachometer. If the results of either of these checks are outside FIGURE 1-6 Examples of two types of laboratory tolerance, then the centrifuge requires service. Some older-model centrifuges. A. Swing-out rotor with buckets and B. centrifuges have carbon brushes in the motor that make contact fixed-angle rotor. with the rotating armature. These brushes wear down in time an  CHAPTER 1 LAboRAToRy bAsiCs 11 require replacement. When these brushes wear down, the cen-trifuge bathbutwillmaintain aconstant temperature within{0.5C.A can no longer maintain speed and will eventually fail to certified thermometer or NIST-calibrated thermometer must be start. Replacing the brushes should be done by a knowledgeable present in the heating block to monitor the temperature. and experienced CLS or someone from the biomedical engineer-ing Heating ovens are used in chromatography procedures to dry department. Refrigerated centrifuges must have temperature chemicals, assist in organic extractions, and dry membranes or checks performed periodically. A NIST-certified thermometer gels used in electrophoresis. Several different designs are available, should be used to verify the temperature of the refrigeration unit. depending on the desired temperature and purpose. Oven designs If the temperature check falls outside acceptable tolerance, then include programmable, vacuum, and standard laboratory types. the unit should be serviced. Temperature control isusually within{1C.Theovenmust have a certified thermometer or NIST-calibrated thermometer available to monitor the interior temperature. WATERBATHS Water baths are routinely used to incubate or warm solutions for a specified period of time. For general clinical laboratory use, water MIxING bathsshould offer variabletemperature control from +5C above Mixing is an operation