Principles of Basic Techniques and Laboratory Safety

Chapter 9


Principles of Basic Techniques and Laboratory Safety



To reliably perform qualitative and quantitative analyses on body fluids and tissue, the clinical laboratorian must understand the basic principles and techniques of analytical chemistry. Valcárcel has generically defined analytical chemistry as “a metrological science that develops, optimizes, and applies measuring processes intended to derive quality analytical information in order to solve the measuring problems posed.”25 Factors that affect the analytical process and operation of the clinical laboratory include knowledge of (1) the concept of solute and solvent; (2) units of measurement; (3) chemicals; (4) reference materials; (5) basic techniques, such as volumetric sampling and dispensing, centrifugation, measurement of radioactivity, gravimetry, thermometry, buffer solution, and processing of solutions; and (6) safety.



Concept of Solute and Solvent


Many analyses in the clinical laboratory are concerned with determination of the presence or measurement of the concentration of substances in solutions, the solutions most often being blood, serum, urine, spinal fluid, or other body fluids (see Chapter 7).


A solution is a homogeneous mixture of one or more solutes dispersed molecularly in a sufficient quantity of a dissolving solvent. In laboratory practice, solutes are typically measured and are frequently referred to as analytes or measurands. A solution may be gaseous, liquid, or solid. A clinical laboratorian is concerned primarily with the measurement of gases or solids in liquids, where there is always a relatively large amount of solvent in comparison with the amount of solute.



Expressing Concentrations of Solutions


The following equations define the expression of concentrations:


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In the past, milliequivalent (mEq) was used to express the concentration of electrolytes in plasma. Now, the recommended unit for expressing the concentration of an electrolyte in plasma is millimoles per liter (mmol/L). For example, if a sample contains 322 mg of Na per deciliter then the serum contains 3220 mg/L. The molar concentration of Na is


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In clinical laboratory practice, a titer is thought of as the lowest dilution at which a particular reaction takes place. Titer is customarily expressed as a ratio, for example, 1:10 or 1 to 10.


Regarding gases in solution, Henry’s law states that the solubility of a gas in a liquid is directly proportional to the pressure of the gas above the liquid at equilibrium. Thus as the pressure of a gas is doubled, its solubility is also doubled. The relationship between pressure and solubility varies with the nature of the gas. When several gases are dissolved at the same time in a single solvent, the solubility of each gas is proportional to its partial pressure in the mixture. The solubility of most gases in liquids decreases with an increase in temperature, and indeed boiling a liquid frequently drives out all dissolved gases. Traditionally, the unit used to describe the concentration of gases in liquids has been percent by volume (vol/vol). Using the SI, gas concentrations are expressed in moles per cubic meter (mol/m3).



Units of Measurement


A meaningful measurement is expressed with both a number and a unit. The unit identifies the dimension—mass, volume, or concentration—of a measured property. The number indicates how many units are contained in the property.


Traditionally, measurements in the clinical laboratory have been made in metric units. In the early development of the metric system, units were referenced to length, mass, and time. The first absolute systems were based on the centimeter, gram, and second (CGS), and then the meter, kilogram, and second (MKS). The Système Internationale d’Unités (SI) is a different system that was accepted internationally in 1960. The units of the system are called SI units.



International System of Units


Base, derived, and supplemental units are the three classes of SI units.20 The eight fundamental base units are listed in Table 9-1. A derived unit is derived mathematically from two or more base units (Table 9-2). A supplemental unit is a unit that conforms to the SI but has not been classified as either base or derived. At present, only the radian (for plane angles) and the steradian (for solid angles) are classified this way.




The Conférence Générale des Poids et Mésures (CGPM) recognizes that some units outside the SI continue to be important and useful in particular applications. An example is the liter as the reference volume in clinical analyses. Liter is the name of the submultiple (cubic decimeter) of the SI unit of volume, the cubic meter. Considering that 1 cubic meter represents some 200 times the blood volume of an adult human, the SI unit of volume is neither a convenient nor a reasonable reference volume in a clinical context. Nevertheless, the CGPM recommends that such exceptional units as the liter should not be combined with SI units and preferably should be replaced with SI units whenever possible.


The minute, hour, and day have had such long-standing use in everyday life that it is unlikely that new SI units derived from the second could supplant them. Some other non-SI units are still accepted, although they are rarely used by most individuals in their daily lives but have been very important in some specialized fields. Details of the SI system are found in an expanded version of this chapter.2


In practical application of units, certain values are too large or too small to be expressed conveniently. Numeric values are brought to convenient size when the unit is appropriately modified by official prefixes (Table 9-3).




Standardized Reporting of Test Results


To describe test results properly, it is important that all necessary information be included in the test description. Systems developed for expressing results produced by the clinical laboratory include the Laboratory Logical Observation Identifiers, Names, and Codes (Lab LOINC),15–17 and the Nomenclature, Properties, and Units (NPU) developed by the International Federation of Clinical Chemistry/International Union of Pure and Applied Chemistry (IFCC/IUPAC).



Lab LOINC System


The Lab LOINC system is a universal coding system for reporting laboratory and other clinical observations to facilitate electronic transmission of laboratory data within and between institutions (http://www.loinc.org). It has several thousand observations in its database. For each observation, there is a code, a long formal name, a short 30-character name, and synonyms. A mapping program termed “Regenstrief LOINC Mapping Assistant” (RELMA) is available to map local test codes to LOINC codes and to facilitate searching of the Lab LOINC database. Both Lab LOINC and RELMA are available at no cost from http://loinc.org/relma (accessed March 16, 2011).




Applications


The Lab LOINC and NPU coding systems are used in context with existing standards, such as the Systematized Nomenclature of Medicine, Clinical Terms (SNOMED CT). Other such coding systems are the ASTM E1238 (American Society for Testing and Materials), HL7 version 2.2 (Health Level Seven; http://www.hl7.org; accessed March 16, 2011), and CEN ENV 1613—a standard developed by the European Committee for Standardization of the Comité Européen de Normalisation (CEN) Technical Committee 251 (http://www.cen.eu/ accessed March 16, 2011).


SNOMED CT is a comprehensive clinical terminology, originally created by the College of American Pathologists (CAP) and, as of April 2007, owned, maintained, and distributed by the International Health Terminology Standards Development Organisation (IHTSDO) in Denmark (http://www.ihtsdo.org; accessed March 16, 2011). IHTSDO is a not-for-profit association that develops and promotes use of SNOMED CT to support safe and effective health information exchange. In practice, the CAP continues to support SNOMED CT operations under contract to the IHTSDO and provides SNOMED-related products and services as a licensee of the terminology.


On April 1, 2009, the owners of LOINC, NPU, and SNOMED CT began an operational trial of prospective divisions of labor in the generation of laboratory test terminology content. This trial will provide practical experience and important information on opportunities to decrease duplication of effort in the development of laboratory test terminology and to ensure that SNOMED CT works effectively in combination with LOINC or NPU.



Chemicals


The quality of the analytical results produced by the laboratory is a direct indication of the purity of the chemicals used as analytical reagents. The availability and quality of the reference materials used to calibrate assays and to monitor their analytical performance also are important.


Laboratory chemicals are available in a variety of grades. The solutes and solvents used in analytical work are reagent grade chemicals, among which water is a solvent of primary importance.



Reagent Grade Water


Preparation of many reagents and solutions used in the clinical laboratory requires “pure” water. Single-distilled water fails to meet the specifications for Clinical Laboratory Reagent Water (CLRW) established by the Clinical Laboratory and Standards Institute (CLSI).10 Because the terms deionized water and distilled water describe preparation techniques, they should be replaced by reagent grade water, followed by the designation of CLRW, which better defines the specifications of the water and is independent of the method of preparation (Table 9-4).




Preparation of Reagent Grade Water


Distillation, ion exchange, reverse osmosis, and ultraviolet oxidation are processes used to prepare reagent grade water. In practice, water is filtered before any of these processes are used.




Ion Exchange

Ion exchange is a process that removes ions to produce mineral-free deionized water. Such water is most conveniently prepared using commercial equipment, which ranges in size from small, disposable cartridges to large, resin-containing tanks. Deionization is accomplished by passing feed water through columns containing insoluble resin polymers that exchange H+ and OH− ions for the impurities present in ionized form in the water. The columns may contain cation exchangers, anion exchangers, or a mixed-bed resin exchanger, which is a mixture of cation- and anion-exchange resins in the same container.


A single-bed deionizer generally is capable of producing water that has a specific resistance in excess of 1 MΩ/cm. When connected in series, mixed-bed deionizers usually produce water with a specific resistance that exceeds 10 MΩ/cm.





Quality, Use, and Storage of Reagent Grade Water


Type III water may be used for glassware washing. (Final rinsing, however, should be done with the water grade suitable for the intended glassware use.) It may also be used for certain qualitative procedures, such as those used in general urinalysis.


Type II water is used for general laboratory testing not requiring type I water. Storage should be kept to a minimum; storage and delivery systems should be constructed to ensure a minimum of chemical or bacterial contamination.


Type I water should be used in test methods requiring minimal interference and maximal precision and accuracy. Such procedures include trace metal, enzyme, and electrolyte measurements, and preparation of all calibrators and solutions of reference materials. This water should be used immediately after production. No specifications for storage systems for type I water are given because it is not possible to maintain high resistivity while drawing off water and storing it.



Testing for Water Purity


At a minimum, water should be tested for microbiological content, pH, resistivity, and soluble silica,10 and the maximum interval in the testing cycle for purity of reagent water should be 1 week. It should be noted that measurements taken at the time of production may differ from those taken at the time and place of use. For example, if the water is piped a long distance, consideration must be given to deterioration en route to the site of use. To meet the specifications for high-performance liquid chromatography (HPLC), in some instances it may be necessary to add a final 0.1-µm membrane filter. The water can be tested by HPLC using a gradient program and monitoring with an ultraviolet (UV) detector. No peaks exceeding the analytical noise of the system should be found.



Reagent Grade or Analytical Reagent Grade (AR) Chemicals


Chemicals that meet specifications of the American Chemical Society (ACS) are described as reagent or analytical reagent grade. These specifications have also become the de facto standards for chemicals used in many high-purity applications. These are available in two forms: (1) lot-analyzed reagents, in which each individual lot is analyzed and the actual amount of impurity reported, and (2) maximum impurities reagents, for which maximum impurities are listed. The Committee on Analytical Reagents of the ACS periodically publishes “Reagent Chemicals” listing specifications (http://pubs.acs.org/reagents/index.html; accessed March 16, 2011). These reagent grade chemicals are of very high purity and are recommended for quantitative or qualitative analyses.



Ultrapure Reagents


Many analytical techniques require reagents whose purity exceeds the specifications of those described previously. Manufacturers offer selected chemicals that have been especially purified to meet specific requirements. There is no uniform designation for these chemicals and organic solvents. Terms such as spectrograde, nanograde, and HPLC pure have been used. Data of interest to the user (e.g., absorbance at a specific UV wavelength) are supplied with the reagent.


Other designations of chemical purity include chemically pure (CP), USP and NF grade [chemicals produced to meet specifications set down in the U.S. Pharmacopeia (USP) or the National Formulary (NF)]. Chemicals labeled purified, practical, technical, or commercial grade should not be used in clinical chemical analysis without prior purification.



Reference Materials


A reference material is a material or substance with one or more physical or chemical properties that is sufficiently well established to be used for (1) calibrating instruments, (2) validating methods, (3) assigning values to materials, and (4) evaluating the comparability of results. Reference materials are of prime importance in establishing metrologic transferability (http://www.bipm.org; accessed March 16, 2011),1,26 a term defined as “the property of a measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty.”13


Primary, secondary, standard, and certified are types of reference materials.





Standard Reference Materials (SRMs)


Standard reference materials (SRMs) for clinical and molecular laboratories are available from the National Institute of Standards and Technology (NIST; http://ts.nist.gov; accessed March 16, 2011). Cholesterol, the first SRM developed by the NIST, was issued in 1967. It should be noted that not all SRMs have the properties and the degree of purity specified for a primary standard, but each has been well characterized for certain chemical or physical properties and is issued with a certificate that gives results of the characterization. These may then be used to characterize other materials.


Examples of SRMs that are available from the NIST for use in clinical and molecular diagnostics laboratories include (1) pure crystalline standards (Table 9-5), (2) human-based standards (Table 9-6), (3) animal blood standards (Table 9-7), (4) standards containing drugs of abuse in urine and human hair (Table 9-8), and (5) SRMs used for DNA profiling/crime scene investigations (Table 9-9).








Certified Reference Materials (CRMs)


Certified reference materials (CRMs) are available for clinical and molecular laboratories from the Institute for Reference Materials and Measurements (IRMM) in Geel, Belgium (http://www.irmm.jrc.be; accessed March 16, 2011). The IRMM is one of the seven institutes of the Joint Research Centre (JRC), a Directorate-General of the European Commission (EC). Other acronyms used to label IRMM reference materials include ERM (European Reference Materials), BCR (Community Bureau of Reference of the Commission of the European Communities), and the IFCC (International Federation of Clinical Chemistry).


Examples of available IRMM standards are listed in Tables 9-10 and 9-11. Reference materials also are available from the World Health Organization (WHO; http://www.who.int/biologicals; accessed March 16, 2011).



TABLE 9-10


Reference Materials Available from the Institute for Reference Materials and Measurements (www.irmm.jrc.be; accessed March 16, 2011)





























































































Number Description
BCR-304 Lyophilized Human Serum
BCR-573; 574; and 575 Creatinine in Human Serum
IRMM-468 and 469 Thyroxine (T4) and Triiodothyronine (T3), Two Levels Each
ERM-DA451/IFCC Cortisol Reference Panel of Fresh Frozen Human Serum
ERM-DA192 and 193 Cortisol in Human Serum
BCR-348R and BCR-DA347 Progesterone in Human Serum
BCR-576; 577; and 578 17-β-Estradiol in Human Serum
ERM-CE-194; 195; and 196 Pb and Cd in Lyophilized Bovine Blood
BCR-634; 635; and 636 Pb and Cd in Lyophilized Human Blood
BCR-637; 638; and 639 Al, Se, and Zn in Human Serum
BCR-393 Lyophilized APO A1 from Human Serum
BCR-457 Human Thyroglobin (Tg)
BCR-486 Purified Alpha Fetoprotein (AFP)
BCR-613 Prostate Specific Antigen (PSA) in the Reconstituted Material
BCR-405 Glycated Hemoglobin (HbA1c) in Human Hemolysate
ERM-DA470k Human Serum Proteins
ERM-DA472/IFCC C-Reactive Protein (CRP)
BCR-522 Hemiglobincyanide (HCN) in Bovine Blood Lysate
IRMM/IFCC-466 and 467 Hemoglobin Isolated from Whole Blood
BCR-410 Prostatic Acid Phosphatase from Human Prostate
BCR-647 Human Adenosine Deaminase (ADA1) from Human Erythrocytes
BCR-693 Human Pancreatic Lipase from Pancreatic Juice
BCR-6974 Human Pancreatic Lipase (Recombinant)
ERM-AD452/IFCC γ-Glutamyltransferase from Pig Kidney
ERM-AD453/IFCC Human Lactate Dehydrogenase Isoenzyme 1
ERM-AD454/IFCC Alanine Aminotransferase from Pig Heart
ERM-AD455/IFCC Creatine Kinase (CK-MB) from Human Heart
IRMM/IFCC-456 Human Pancreatic α-Amylase
ERM-AD457/IFCC Aspartate Transaminase (AST)


TABLE 9-11


Standards Certified for DNA Sequence Available from the Institute for Reference Materials and Measurements (www.irmm.jrc.be; accessed March 16, 2011)*















Number Plasmid DNA
IRMM/IFCC-490 Sequence of 609 bp DNA Fragment from Human Prothrombin Gene (G20210 Wildtype Sequence)
IRMM/IFCC-491 Sequence of 609 bp DNA Fragment from Human Prothrombin Gene (Point Mutation G20210A)
IRMM/IFCC-492 Sequence of 609 bp DNA Fragment from Human Prothrombin Gene (G20210 Wildtype and Point Mutation G20210A Sequences)

bp, Base pairs.


*Availability: Each polypropylene vial contains approximately 1 ng plasmid DNA in a volume of 50 µL of a tris/EDTA solution.



Basic Techniques and Procedures


Basic practices used in clinical and molecular diagnostic laboratories include (1) optical, (2) chromatographic, (3) electrochemical, (4) electrophoretic, (5) mass spectrometric, (6) enzymatic, and (7) immunoassay techniques. These techniques are discussed in detail in Chapters 10 through 16. Here we discuss the basic techniques of volumetric sampling and dispensing, centrifugation, measurement of radioactivity, gravimetry, thermometry, controlling hydrogen ion concentration, and processing solutions.



Volumetric Sampling and Dispensing


Clinical chemistry procedures require accurate volumetric measurements to ensure accurate results. For accurate work, only Class A glassware should be used. Class A glassware is certified to conform to the specifications outlined in NIST circular C-602.



Pipettes


Pipettes are used for the transfer of a volume of liquid from one container to another. They are designed either (1) to contain (TC) a specific volume of liquid or (2) to deliver (TD) a specified volume. Pipettes used in clinical, molecular diagnostic, and analytical laboratories include (1) manual transfer and measuring pipettes, (2) micropipettes, and (3) electronic and mechanical pipetting devices. Developments in improved design of pipetting systems include robotic automation, the capability to provide electronic and personal computer (PC) control of pipetting devices, and careful attention to advanced ergonomic design features. Automatic photometric pipette calibration systems are also available that reduce the time needed to periodically check pipettes and potentially allow more efficient use of personnel.



Transfer and Measuring Pipettes

A transfer pipette is designed to transfer a known volume of liquid. Measuring and serologic pipettes are scored in units such that any volume up to a maximum capacity is delivered.



Transfer Pipettes: Transfer pipettes include both volumetric and Ostwald-Folin pipettes (Figure 9-1). They consist of a cylindrical bulb joined at both ends to narrower glass tubing. A calibration mark is etched around the upper suction tube, and the lower delivery tube is drawn out to a gradual taper. The bore of the delivery orifice should be sufficiently narrow to allow rapid outflow of liquid and incomplete drainage cannot cause measurement errors beyond tolerances specified.



A volumetric transfer pipette (Figure 9-1, A) is calibrated to deliver accurately a fixed volume of a dilute aqueous solution. The reliability of the calibration of the volumetric pipette decreases with decreased size, and therefore special micropipettes have been developed.


Ostwald–Folin pipettes (Figure 9-1, B) are similar to volumetric pipettes but have the bulb closer to the delivery tip and are used for accurate measurement of viscous fluids, such as blood or serum. In contrast to a volumetric pipette, an Ostwald-Folin pipette has an etched ring near the mouthpiece, indicating that it is a blow-out pipette. With the use of a pipetting bulb, the liquid is blown out of the pipette only after the blood or serum has drained to the last drop in the delivery tip. When filled with opaque fluids, such as blood, the top of the meniscus must be read. Controlled slow drainage is required with all viscous solutions so that no residual film is left on the walls of the pipette.



Measuring Pipettes: The second principal type of pipette is the graduated or measuring pipette (Figure 9-1, C). This is a piece of glass tubing that is drawn out to a tip and graduated uniformly along its length. Two types are available. The Mohr pipette is calibrated between two marks on the stem, whereas the serologic pipette has graduated marks down to the tip. The serologic pipette (Figure 9-1, D) must be blown out to deliver the entire volume of the pipette and has an etched ring (or pair of rings) near the bulb end of the pipette signifying that it is a blow-out pipette. Mohr pipettes require controlled delivery of the solution between the calibration marks. Serologic pipettes have a larger orifice than do Mohr pipettes and thus drain faster. In practice, measuring pipettes are used principally for measurement of reagents and generally are not considered sufficiently accurate for measuring samples and calibrators.

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