CHAPTER 1 © Yurchanka Siarhei/Shutterstock.

Basic Principles and

Practices of Clinical
Chemistry
Kathryn Dugan and Elizabeth Warning

CHAPTER OUTLINE
Units of Measure Laboratory Mathematics and Calculations
Reagents Significant Figures
Chemicals Logarithms
Reference Materials Concentration
Water Specifications Dilutions
Solution Properties Simple Dilutions
Concentration Serial Dilutions
Colligative Properties Water of Hydration
Redox Potential Graphing and Beer’s Law
Conductivity Specimen Collection and Handling
pH and Buffers Types of Samples
Laboratory Equipment Sample Processing
Heating Units Sample Variables
Glassware and Plasticware Chain of Custody
Desiccators and Desiccants Electronic and Paper Reporting of Results
Balances References
Centrifuges

KEY TERMS
Analyte Density Hygroscopic
Anhydrous Desiccant Icterus
Arterial blood Dilution International unit
Beer’s law Distilled water Ionic strength
Buffer Equivalent weight Linearity
Calibration Erlenmeyer flasks Lipemia
Centrifugation Filtration Molality
Cerebrospinal fluid (CSF) Graduated cylinder Molarity
Colligative property Griffin Beaker Normality
Conductivity Hemolysis One-point calibration
Deionized water Henderson-Hasselbalch equation Osmotic pressure
Delta absorbance Hydrate Oxidized
2
 Basic Principles and Practices of Clinical Chemistry 3

Oxidizing agent Reducing agent Specific gravity

Percent solution Reverse osmosis Standard reference materials
pH Serial dilution (SRMs)
Pipette Serum Système International d’Unités (SI)
Primary standard Significant figures Thermistor
Reagent-grade water Solute Valence
Redox potential Solution Volumetric
Reduced Solvent Whole blood

CHAPTER OBJECTIVES
Upon completion of this chapter, the clinical laboratorian • List and describe the types of thermometers used in
should be able to do the following: the clinical laboratory.
• Classify the type of pipette when given an actual
• Convert results from one unit format to another using
pipette or its description.
the SI and traditional systems.
• Demonstrate the proper use of a measuring and
• Describe the classifications used for reagent-grade
volumetric pipette.
water.
• Describe two ways to calibrate a pipetting device.
• Identify the varying chemical grades used in reagent
• Define a desiccant and discuss how it is used in the
preparation and indicate their correct use.
clinical laboratory.
• Define primary standard and standard reference
• Describe how to properly care for and balance a
materials.
centrifuge.
• Describe the following terms that are associated
• Correctly perform the laboratory mathematical
with solutions and, when appropriate, provide the
calculations provided in this chapter.
respective units: percent, molarity, normality, molality,
• Identify and describe the types of samples used in
saturation, colligative properties, redox potential, and
clinical chemistry.
conductivity.
• Outline the general steps for processing blood
• Compare and contrast osmolarity and osmolality.
samples.
• Define a buffer and give the formula for pH and pK
• Apply Beer’s law to determine the concentration
calculations.
of a sample when the absorbance or change in
• Use the Henderson-Hasselbalch equation to
absorbance is provided.
determine the missing variable when given either the
• Identify the preanalytic variables that can adversely
pK and pH or the pK and concentration of the weak
affect laboratory results as presented in this chapter.
acid and its conjugate base.

CASE STUDY 1.1, PART 1 CASE STUDY 1.2, PART 1

Meet Miles, a 25-year-old grad- Meet Mía, a 35-year-old grad-
uate who accepted his first job uate who is also newly hired
offer working in the chemistry and works as a generalist in
department at a large medi- a small community hospi-
cal center. Miles and Mía were tal. Mía received a rainbow
classmates in college and often of tubes from the emergency
support each other on technical department. She handed her
issues, even though they work coworker the lavender- and
at different facilities within the blue-top tubes and placed the
same health system. 8.0-mL plain red-top tube and
© dotshock/Shutterstock.
the 3.5-mL plasma separa- © Ariel Skelley/DigitalVision/Getty Images.
tor tube in the centrifuge. She
placed the heparinized whole
blood specimen on the mixer and logged in to the labora-
tory information system to receive the specimens. Once
the specimens were accessioned, she ran a STAT profile
on the Nova pHOx analyzer using the whole blood speci-
men, and the results were autoverified.
4 Chapter 1 Basic Principles and Practices of Clinical Chemistry

The primary purpose of a clinical chemistry laboratory Table 1.1 SI Units

is to perform analytic procedures that yield a­ ccurate
and precise information, aiding in patient diagnosis Base Quantity Name Symbol
and treatment. The achievement of reliable results Length Meter m
requires that the clinical laboratorian be able to cor-
Mass Kilogram kg
rectly use basic supplies and equipment and possess
an understanding of fundamental concepts critical Time Second s
to any analytic procedure. The topics in this chapter Electric current Ampere A
include units of measure, basic laboratory supplies,
and introductory laboratory mathematics, plus a brief Thermodynamic Kelvin K
discussion of specimen collection, processing, and temperature
reporting. Amount of Mole mol
substance

Units of Measure Luminous intensity Candela cd

Selected Derived
Any meaningful quantitative laboratory result consists
of two components: the first component represents Frequency Hertz Hz
the number related to the actual test value, and the Force Newton N
second is a label identifying the units. The unit defines
the physical quantity or dimension, such as mass, Celsius temperature Degree Celsius °C
length, time, or volume.1 There are a few laboratory Catalytic activity Katal kat
tests that do not have units, but whenever possible,
units should be used. Selected Accepted Non-SI
The Système International d’Unités (SI) was Minute (time) (60 s) min
adopted in 1960. It is preferred in scientific litera-
Hour (3600 s) h
ture and clinical laboratories and is the only system
employed in many countries. This system was devised Day (86,400 s) d
to provide the global scientific community with a uni- Liter (volume) L
(1 dm3 = 10–3 m3)
form method of describing physical quantities. The SI
system units (referred to as SI units) are based on the Angstrom (0.1 nm = 10–10 m) Å
metric system. Several subclassifications exist within
© Jones & Bartlett Learning.
the SI system, one of which is the basic unit. There
are seven basic units (Table 1.1), with length (meter),
mass (kilogram), and quantity of a substance (mole)
being the units most frequently encountered. Derived or expressed in scientific notation as 1 × 103 L.
units are another subclassification of the SI system. Table 1.2 indicates prefixes that are frequently used in
A derived unit is a mathematical function describing clinical laboratories. Prefixes smaller than the basic unit
one of the basic units. An example of an SI-derived have a negative exponent (deci: 10–1), and prefixes
unit is meters per second (m/s), which is used to larger than the base unit have a positive exponent
express velocity. Some non-SI units are so widely used (kilo: 103). When converting between prefixes, note
that they have become acceptable for use within the SI the relationship between the two prefixes based on
system (Table 1.1). These include units such as hour, whether you are changing to a smaller or larger prefix.
minute, day, gram, liter, and plane angles expressed When converting from a larger to smaller, the decimal
as degrees. The SI system uses standard prefixes to will move to the right. For example, converting one
­indicate a decimal fraction or multiples of that basic liter (1.0 × 100 or 1.0) to milliliters (1.0 × 10−3 or
unit (Table 1.2).1 For example, 0.001 liter can be 0.001), the starting unit (L) is larger than ­milliliters,
expressed using the prefix milli, or 10–3, and since it by a factor of 1000, or 103. This means that the
requires moving the decimal point three places to the decimal place moves to the right three places, so
right, it can then be written as 1 milliliter, or abbrevi- 1.0 liter (L) equals 1000 milliliters (mL). The oppo-
ated as 1 mL. It may also be written in scientific nota- site is also true. When converting to a larger unit, the
tion as 1 × 10–3 L. Likewise, 1000 liters would use the decimal place moves to the left. For example, convert-
prefix of kilo (103) and could be written as 1 k ­ iloliter ing 1000 milliliters (mL) to 1.0 liter (L), the decimal
 Units of Measure 5

Example 1: Convert 1.0 L to μL

SI CONVERSIONS
To convert between SI units, move the decimal the
1.0 L (1 × 100)
difference between the exponents represented by μL (micro = 10–6)
the prefix of the base unit. When moving from a
larger unit to a smaller unit, the decimal will move The difference between the exponents = 6. The con-
to the right. When converting from a smaller unit to version is from a larger unit to a smaller unit, so the
a larger unit, the decimal will move to the left. decimal will move 6 places to the right.
If converting from smaller unit to larger
unit, then move decimal to the left the exponent 1.0 L = 1,000,000 μL
difference.
If converting from larger unit to smaller unit,
then move decimal to the right the exponent Example 2: Convert 5 mL to μL
difference.
5 mL (milli = 10−3)
μL (micro = 10−6)
point moves to the left three places to become 1.0 L. The difference between the exponents = 3. The con-
Note that the SI term for mass is kilogram, which is version is from a larger unit to a smaller unit, so the
the only basic unit that contains a prefix as part of its decimal will move 3 places to the right.
name. Generally, the clinical laboratory uses the term
gram for mass rather than kilogram. 5 mL = 5000 μL

Table 1.2 Prefixes Used with SI Units

Factor Prefix Symbol
10−18 atto a 0.000000000000000001
10−15 femto f 0.000000000000001
10−12 pico p 0.000000000001
10−9 nano n 0.000000001
10−6 micro μ 0.000001
10−3 milli m 0.001
10–2 centi c 0.01
10–1 deci d 0.1
100 Liter, meter, gram Basic unit 1.0
101 deca da 10
102 hecto h 100
103 kilo k 1000
10 6
mega M 1,000,000
109 giga G 1,000,000,000
1012 tera T 1,000,000,000,000
1015 peta P 1,000,000,000,000,000
1018 exa E 1,000,000,000,000,000,000

Prefixes are used to indicate a subunit or multiple of a basic SI unit.

© Jones & Bartlett Learning.
6 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Example 3: Convert 5.3 mL to dL Chemicals

5.3 mL (milli = 10−3) Analytic chemicals exist in varying grades of purity:
dL (deci = 10−1) Reagent grade or analytic reagent (AR); ultrapure,
chemically pure (CP); United States Pharmacopeia
The conversion is moving from a smaller unit to a (USP); National Formulary (NF); and technical or
larger unit, so the decimal place will move two places commercial grade.3 Chemicals with AR designation
to the left. are suitable for use in most analytic laboratory proce-
dures. A committee of the American Chemical Society
5.3 mL = 0.053 dL (ACS) established specifications for AR grade chem-
icals, and chemical manufacturers must either meet
Reporting of laboratory results is often expressed
or exceed these requirements. The labels on reagents
in terms of substance concentration (e.g., moles) should clearly state the actual impurities for each
or the mass of a substance (e.g., mg/dL, g/dL, chemical lot or list the maximum allowable impu-
g/L, mmol/L, and IU) rather than in SI units. These rities. The label should also include one of the fol-
traditional units can cause confusion during inter- lowing designations: AR or ACS or For laboratory use
pretation and conversion to SI units: examples of or ACS Standard-Grade Reference Materials. Ultrapure
conversions can be found later in the chapter. As
­ chemicals have additional purification steps for use in
with other areas of industry, the laboratory and specific procedures such as chromatography, immu-
the rest of medicine are moving toward adopt- noassays, molecular diagnostics, standardization, or
ing universal standards promoted by the Inter- other techniques that require extremely pure chem-
national Organization for Standardization, often icals. These reagents may have designations of HPLC
referred to as ISO. This group develops standards (high-performance liquid chromatography) or chro-
of practice, definitions, and guidelines that can matographic on their labels.
be adopted by everyone in a given field, provid- Because USP- and NF-grade chemicals are used to
ing for more uniform terminology. Many national manufacture drugs, the limitations established for this
initiatives have recommended common units for group of chemicals are based only on the criterion of
laboratory test results, but none have been widely not being injurious to individuals. Chemicals in this
adopted.2 As with any transition, the clinical labo- group may be pure enough for use in most chemical
ratorian should be familiar with all the terms cur- procedures, but the purity standards they meet are
rently used in their field and how to convert these to not based on the needs of the laboratory and may or
SI units. may not meet all assay requirements.
Reagent designations of CP or ultrapure grade
indicate that the impurity limitations are not stated,
Reagents and preparation of these chemicals is not uniform.
It is not recommended that clinical laboratories use
In today’s highly automated laboratory, there is lit- these chemicals for reagent preparation unless further
tle need for reagent preparation by the laboratorian. purification or a reagent blank is included. Technical
Most instrument manufacturers make the reagents or commercial grade reagents are used primarily in
in a ready-to-use form or “kit” in which all neces- manufacturing and should never be used in the clin-
sary reagents and respective storage containers are ical laboratory.
prepackaged as a unit, requiring only the addition Organic reagents also have varying grades of
of water or buffer for reconstitution. A heightened purity that differ from those used to classify inor-
awareness of the hazards of certain chemicals and ganic reagents. These grades include a practical
the numerous regulatory agency requirements has grade with some impurities; CP, which approaches
caused clinical chemistry laboratories to eliminate the purity level of reagent-grade chemicals; spectro-
massive stocks of chemicals and opt instead for scopic (spectrally pure) and chromatographic grade
the ease of using prepared reagents. Periodically, organic reagents; and reagent grade (ACS), which
the laboratorian may still need to prepare reagents is certified to contain impurities below established
or solutions, especially in hospital laboratories ACS levels. Other than the purity aspects of the
involved in research and development, biotechnol- chemicals, laws related to the Occupational Safety
ogy applications, specialized analyses, or method and Health Administration (OSHA)4 require manu-
validation. facturers to indicate any physical or biologic health
 Reagents 7

hazards and precautions needed for the safe use, Water Specifications8
storage, and disposal of any chemical. Manufacturers
Water is the most frequently used reagent in the
are required to provide a Safety Data Sheet (SDS). A
laboratory. Tap water is unsuitable for laboratory
copy of the SDS must be readily available to ensure
applications. Most procedures, including reagent
the safety of laboratorians.
and control preparation, require water that has been
substantially purified, known as reagent-grade
Reference Materials water. There are various water purification methods
Unlike other areas of chemistry, clinical chemistry is including ­distillation, ion exchange, reverse osmo-
involved in the analysis of biochemical by-products sis, ultrafiltration, ultraviolet light, sterilization, and
found in biological fluids, such as serum, plasma, or ozone treatment. According to the Clinical and Lab-
urine. For this reason, traditionally defined standards oratory Standards Institute (CLSI), reagent-grade
used in analytical chemistry do not readily apply in water is classified into one of six categories based
clinical chemistry. on the specifications needed for its use rather than
A primary standard is a highly purified chem- the method of purification or preparation.9 These
ical that can be measured directly to have an exact categories include clinical laboratory reagent water
known concentration and purity. The ACS has purity (CLRW), special reagent water (SRW), instrument
tolerances for primary standards; because most bio- feed water, water supplied by method manufacturer,
logic constituents are unavailable within these toler- autoclave and wash water, and commercially bottled
ance limitations, the National Institute of ­Standards purified water. Each category has a specific accept-
and Technology (NIST) has certified standard able limit. The College of American Pathologists
­reference materials (SRMs) that are used in place requires laboratories to define the specific type of
of ACS primary standard materials.5–7 water required for each of its testing procedures and
These SRMs are assigned a value after analysis requires water quality testing at least annually. Water
using state-of-the-art methods and equipment. The quality testing routinely includes monitoring micro-
chemical composition of these substances is then bial colony-forming units/mL and may also include
certified; however, they may not have the purity of a other parameters.
primary standard. Because each substance has been Distilled water has been purified to remove
characterized for certain chemical or physical prop- almost all organic materials, using a technique of
erties, it can be used in place of an ACS primary distillation where water is boiled and vaporized.
standard in clinical work and is often used to verify Many impurities do not rise in the water vapor and
­ alibration or accuracy/bias assessments. Many
c will remain in the boiling apparatus so that the water
manufacturers use a NIST SRM when producing cal- collected after condensation has less contamination.
ibrator and standard materials. These materials are Water may be distilled more than once, with each
considered “traceable to NIST” and may meet certain distillation cycle removing additional impurities.
accreditation requirements. Standard reference Ultrafiltration and nanofiltration, like distillation,
materials are used for l­ inearity studies to determine are excellent in removing particulate matter, micro-
the relationship between the standard’s concentra- organisms, and any pyrogens or endotoxins.
tion and the instrument result. Linearity studies Deionized water has some or all ions removed,
are required when a new test or new test method- although organic material may still be present, so it is
ology is introduced. There are SRMs for a number neither pure nor sterile. Generally, deionized water is
of routine analytes, hormones, drugs, and blood purified from previously treated water, such as prefil-
gases, with others being added.5 Calibration of an tered or distilled water. Deionized water is produced
instrument is a process that pairs an analytical sig- using either an anion- or a cation-exchange resin,
nal with a concentration value of an analyte. When followed by replacement of the removed ions with
performing a calibration, a series of calibrators with hydroxyl or hydrogen ions. A combination of sev-
known concentrations of a specific analyte are used. eral ion-exchange resins will produce different grades
The instrument is programmed with the known of deionized water. A two-bed system uses an anion
concentrations and will adjust the analytic signal resin followed by a cation resin. The different resins
to match the given concentration. Calibrators can may be in separate columns or in the same column.
be purchased as a kit or made by diluting a known This process is excellent at removing dissolved ion-
stock solution. ized solids and dissolved gases.
8 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Reverse osmosis is a process that uses pressure This one measurement does not suffice for determi-
to force water through a semipermeable membrane, nation of true water purity because a nonionic con-
producing a filtered product. Reverse osmosis may be taminant may be present that will have little effect on
used for the pretreatment of water, however, it does resistance. Reagent water meeting specifications from
not remove dissolved gases. other organizations, such as the American Society for
Filtration can remove particulate matter from Testing and Materials (ASTM), may not be equivalent
municipal water supplies before any additional treat- to those established by the CLSI, so care should be
ments. Filtration cartridges can be composed of glass, taken to meet the assay procedural requirements for
cotton, or activated charcoal, which removes organic water type.
materials and chlorine. Some have submicron filters
(≤0.2 µm), which remove any substances larger than Solution Properties
the filter’s pores, including bacteria. The use of these fil-
In clinical chemistry, substances found in biologic flu-
ters depends on the quality of the municipal water and
ids, including serum, plasma, urine, and spinal fluid,
the other purification methods used. For example, hard
are quantified. A substance that is dissolved in a liq-
water (containing calcium, iron, and other dissolved
uid is called a solute; a biologic solute is also known
elements) may require prefiltration with a glass or cot-
as an analyte. The liquid in which the solute is dis-
ton filter rather than activated charcoal or submicron
solved—for example, a biologic fluid—is the solvent.
filters, which quickly become clogged and are expensive
Together, solute and solvent represent a solution. Any
to use. The submicron filter may be better suited after
chemical or biologic solution can be described by its
distillation, deionization, or reverse osmosis treatment.
basic properties, including concentration, saturation,
Ultraviolet oxidation, which removes some trace
colligative properties, redox potential, conductivity,
organic material or sterilization processes at specific
density, pH, and ionic strength.
wavelengths, can destroy bacteria when used as part
of a system but may leave behind some residual prod-
ucts. This technique is often followed by other purifi- Concentration
cation processes. The analyte concentration in solution can be expressed
Reagent-grade water can be obtained by initially in many ways. Concentration is commonly expressed
filtering to remove particulate matter, followed by as percent solution, molarity, molality, or normality. These
reverse osmosis, deionization, and a 0.2-µm filter or are non-SI units, however; the SI unit for the amount
more restrictive filtration process. Autoclave wash of a substance is the mole. Examples of concentration
water is acceptable for glassware washing but not for calculations are provided later in this chapter.
analysis or reagent preparation. SRW is used for spe- Percent solution is expressed as the amount of
cific techniques like the HPLC, molecular diagnostics, solute per 100 total units of solution. Three expres-
or mass spectrophotometry, which may require spe- sions of percent solutions are weight per weight
cific parameters for the analysis. All SRW should meet (w/w), volume per volume (v/v), and weight per vol-
CLRW standards and, depending on the application, ume (w/v). Weight per weight (% w/w) refers to the
CLRW should be stored in a manner that reduces any number of grams of solute per 100 g of solution. Vol-
chemical or bacterial contamination and for short ume per volume (% v/v) is used for liquid solutes and
periods. gives the milliliters of solute in 100 mL of solution.
Testing procedures to determine the quality of For v/v solutions, it is recommended that grams per
reagent-grade water include measurements of resis- deciliter (g/dL) be used instead of % v/v. Weight per
tance, pH, colony counts on selective and nonselective volume (% w/v) is the most commonly used percent
media for the detection of bacterial contamination, solution in the clinical laboratory and is defined as
chlorine, ammonia, nitrate or nitrite, iron, hardness, the number of grams of solute in 100 mL of solution.
phosphate, sodium, silica, carbon dioxide, chemical Weight per volume is not the same as molarity, and
oxygen demand, and metal detection. Some accred- care must be taken to not confuse the two. Examples
itation agencies10 recommend that laboratories docu- of percent solution calculations can be found later in
ment culture growth, pH, and specific resistance on this chapter.
water used in reagent preparation. Resistance is mea- Molarity (M) is expressed as the number of moles
sured because pure water, devoid of ions, is a poor per 1 L of solution. One mole of a substance equals
conductor of electricity and has increased resistance. its gram molecular weight (gmw), so the customary
The relationship of water purity to resistance is lin- units of molarity (M) are moles/liter. The SI represen-
ear; generally, as purity increases, so does resistance. tation for the traditional molar concentration is moles
 Reagents 9

of solute per volume of solution, with the volume of A dilute solution is one in which there is relatively little
the solution given in liters. The SI expression for con- solute or one that has a lower solute concentration
centration should be represented as moles per liter per volume of solvent than the original, such as when
(mol/L), millimoles per liter (mmol/L), micromoles making a dilution. In contrast, a concentrated solution
per liter (μmol/L), or nanomoles per liter (nmol/L). has a large quantity of solute in solution. A solution in
The common concentration term molarity is not an SI which there is an excess of undissolved solute particles
unit for concentration. Molarity depends on volume, can be referred to as a saturated solution. As the name
and any significant physical changes that influence implies, a supersaturated solution has an even greater
volume, such as changes in temperature and pressure, concentration of undissolved solute particles than a
will also influence molarity. Calculations can be found saturated solution of the same ­substance. Because of
in the Laboratory Mathematics and Calculations sec- the greater concentration of solute particles, a super-
tion of this chapter. saturated solution is thermodynamically unstable.
Molality (m) represents the amount of solute The addition of a crystal of solute or mechanical agita-
per 1 kg of solvent. Molality is sometimes confused tion disturbs the supersaturated solution, resulting in
with molarity; however, it can be easily distinguished crystallization of any excess material out of solution.
because molality is always expressed in terms of moles An example is when measuring serum osmolality by
per kilogram (weight per weight) and describes moles freezing point depression.
per 1000 g (1 kg) of solvent. Note that the common
abbreviation (m) for molality is a lowercase “m,” while
the uppercase “M” refers to molarity. Molality is not Colligative Properties
influenced by temperature or pressure because it is Colligative properties are those properties related
based on mass rather than volume. to the number of solute particles per solvent mol-
Normality is the least likely of the four concen- ecules, not on the type of particles present. The
tration expressions to be encountered in clinical lab- behavior of particles or solutes in solution demon-
oratories, but it is often used in chemical titrations strates four properties: osmotic pressure, vapor
and chemical reagent classification. It is defined as the pressure, freezing point, and boiling point. These
number of gram equivalent weights per 1 L of solu- are called c­ olligative properties. Osmotic pressure
tion. An equivalent weight is equal to the gmw of is the pressure that opposes osmosis when a solvent
a substance divided by its valence. The valence is flows through a semipermeable membrane to estab-
the number of units that can combine with or replace lish equilibrium between compartments of differing
1 mole of hydrogen ions for acids and hydroxyl ions concentration. Vapor pressure is the pressure exerted
for bases and the number of electrons exchanged in by the vapor when the liquid solvent is in equilib-
oxidation–reduction reactions. Normality is always
­ rium with the vapor. Freezing point is the temperature
equal to or greater than the molarity of the compound. at which the first crystal (solid) of solvent forms in
Calculations can be found later in this chapter. Nor- equilibrium with the solution. Boiling point is the tem-
mality was previously used for reporting e­lectrolyte perature at which the vapor pressure of the solvent
values, expressed as milliequivalents per liter (mEq/L); reaches atmospheric pressure (usually 1 ­atmosphere).
however, this convention has been replaced with mil- The osmotic pressure of a dilute solution is
limoles per liter (mmol/L). The College of American directly proportional to the concentration of the
Pathologists (CAP) currently requires chloride to molecules in solution. The expression for concen-
be reported in mmol/L. Because the four main elec- tration is the osmole. One osmole of a substance
trolytes, Na+, K+, CO2– (HCO3–), and Cl–, all have a equals the molarity or molality multiplied by the
valence of 1, the concentration reported will remain number of particles, not the kind of particle, at dis-
the same whether the unit is mEq/L or mmol/L. sociation. If molarity is used, the resulting expres-
Solution saturation gives little specific informa- sion would be termed osmolarity; if molality is used,
tion about the concentration of solutes in a solution. the expression changes to osmolality. Osmolality is
A solution is considered saturated when no more sol- preferred since it depends on the weight rather than
vent can be dissolved in the solution. Temperature, as volume and is not readily influenced by temperature
well as the presence of other ions, can influence the and pressure changes. When a solute is dissolved in
solubility constant for a solute in a given solution and a solvent, the colligative properties change in a pre-
thus affect the saturation. Routine terms in the clin- dictable manner for each osmole of substance pres-
ical laboratory that describe the extent of saturation ent. In the clinical setting, freezing point and vapor
are dilute, concentrated, saturated, and supersaturated. pressure depression can be measured as a function
10 Chapter 1 Basic Principles and Practices of Clinical Chemistry

of osmolality. Freezing point is preferred since vapor weak acid or base solution (like a buffer) tends to be
pressure measurements can give inaccurate readings very small, meaning little dissociation occurs.
when some substances, such as alcohols, are present The dissociation of acetic acid (CH3COOH), a
in the samples. weak acid, can be illustrated as follows:

HA A– + H+
Redox Potential (Eq. 1.2)
Redox potential, or oxidation–reduction potential, is a CH 3 COOH CH 3 COO– + H+
measure of the ability of a solution to accept or donate
HA = weak acid, A− = conjugate base, H+ = hydro-
electrons. Substances that donate electrons are called
gen ions, [] = concentration of item in the bracket.
reducing agents; those that accept electrons are
Sometimes, the conjugate base (A−) will be referred
considered oxidizing agents. The ­mnemonic—LEO
to as a “salt” since, physiologically, it will be associated
(lose electrons oxidized) the lion says GER (gain
with some type of cation, such as sodium (Na+).
electrons reduced)—may prove useful when trying
The dissociation constant, Ka, for a weak acid may
to recall the relationship between reducing/oxidizing
be calculated using the following equation:
agents.
A– H+
Ka = (Eq. 1.3)
Conductivity HA
Conductivity is a measure of how well electricity
passes through a solution. A solution’s conductivity Rearrangement of this equation reveals
quality depends principally on the number of respec-
tive charges of the ions present. Resistivity, the recip- HA (Eq. 1.4)
H+ = K a ×
rocal of conductivity, is a measure of a substance’s A+
resistance to the passage of electrical current. The
primary application of resistivity in the clinical labo- Taking the log of each quantity and then multiplying
ratory is for assessing the purity of water. Resistivity by minus 1 (−1), the equation can be rewritten as
(resistance) is expressed as ohms and conductivity is
HA
expressed as ohms−1. – log H+ = – log K a × – log (Eq. 1.5)
A–
pH and Buffers By convention, lowercase p means “negative log of”;
Buffers are weak acids or bases and their related therefore, –log[H+] may be written as pH, and −Ka
salts that minimize changes in the hydrogen ion may be written as pKa. The equation now becomes
concentration. Hydrogen ion concentration is often
HA
expressed as pH. A lowercase p in front of c­ ertain pH = pK a – log (Eq. 1.6)
letters or abbreviations operationally means the A–
“negative logarithm of” or “inverse log of” that
substance. In keeping with this convention, the
­ Eliminating the minus sign in front of the log of the
term pH represents the negative or inverse log of HA
the hydrogen ion concentration. Mathematically, pH quantity results in an equation known as the
A–
is expressed as
Henderson-Hasselbalch equation, which mathe-
1 matically describes the dissociation characteristics of
pH = log weak acids (pKa) and bases (pKb) and the effect on pH:
H+ (Eq. 1.1)

pH = – log H+ A–
pH = pK a + log (Eq. 1.7)
HA
where [H+] equals the concentration of hydrogen
ions in moles per liter (M). The pH scale ranges from When the ratio of [A−] to [HA] is 1, the pH equals the
0 to 14 and is a convenient way to express hydrogen pK and the buffer has its greatest buffering capacity.
ion concentration. The dissociation constant Ka, and therefore the pKa,
Unlike a strong acid or base, which dissociates remains the same for a given substance. Any changes
almost completely, the dissociation constant for a in pH are solely due to the ratio of conjugate base [A−]
 Laboratory Equipment 11

concentration to weak acid [HA] concentration. Refer enzyme determinations, require precise temperature
to Chapter 12, Blood Gases, pH, and Buffer Systems, for control, whereas others work well over a wide range
more information. of temperatures. Reactions that are temperature
Ionic strength is another important aspect of dependent use some type of heating/cooling cell,
buffers, particularly in separation techniques. Ionic heating/cooling block, or water/ice bath to provide
strength is the concentration or activity of ions in a the correct temperature environment. Laboratory
solution or buffer. Increasing ionic strength increases refrigerator temperatures are often critical and need
the ionic cloud surrounding a compound and periodic verification. Thermometers can be an inte-
decreases the rate of particle migration. It can also gral part of an instrument or need to be placed in
promote compound dissociation into ions effectively the device for temperature maintenance and mon-
increasing the solubility of some salts, along with itoring. Several types of temperature devices are
changes in current, which can also affect electropho- currently used in the clinical laboratory, including
retic separation. liquid-in-glass and electronic (thermistor) devices.
Regardless of which type is being used, all tempera-
ture-reading devices must be calibrated for accu-
Laboratory Equipment racy. Liquid-in-glass thermometers use a colored
In today’s clinical chemistry laboratory, there are many liquid (red or other colored material), encased in
different types of equipment in use. Most manual plastic or glass, measuring temperatures between
techniques have been replaced by automation, but it 20°C and 400°C. Visual inspection of the liquid-
is still necessary for the laboratorian to be knowledge- in-glass thermometer should reveal a continuous
able in the operation and use of common laboratory line of liquid, free from separation or bubbles. If
equipment. The following is a brief discussion of the separation or bubbles are present, then replace the
composition and general use of common equipment thermometer.
found in a clinical chemistry laboratory, including Liquid-in-glass thermometers should be cal-
heating units, thermometers, pipettes, flasks, beakers, ibrated against a NIST-certified or NIST-traceable
balances, and centrifuges. thermometer for critical laboratory applications.11
NIST has an SRM thermometer with various calibra-
tion points (0°C, 25°C, 30°C, and 37°C) for use with
Heating Units
­liquid-in-glass thermometers. Gallium, another SRM,
Heat blocks and water baths are common heating has a known melting point and can also be used for
units within the laboratory. The temperature of these thermometer verification.
heating units must be monitored daily when in use. As automation advances and miniaturizes,
The predominant practice for temperature measure- the need for an accurate, fast-reading electronic
ment uses the Celsius (°C) scale; however, Fahr- thermometer (thermistor) has increased and is
enheit (°F) and Kelvin (°K) scales are also used.11 now routinely incorporated in many devices. The
The SI ­designation for temperature is the Kelvin scale. advantages of a thermistor over the more tradi-
Table 1.3 gives the conversion formulas between
tional ­ liquid-in-glass thermometers are size and
­Fahrenheit and Celsius scales, and Appendix C (found ­millisecond response time. Similar to the liquid-in-
in the Navigate 2 digital component) lists the various
glass thermometers, the thermistor can be calibrated
conversion formulas.
against an SRM thermometer.
All analytic reactions occur at an optimal tem-
perature. Some laboratory procedures, such as
Glassware and Plasticware
Until recently, laboratory supplies (e.g., pipettes,
Table 1.3 Common Temperature Conversions flasks, beakers) consisted of some type of glass
Celsius (Centigrade) °C (9/5) + 32 (multiply Celsius and could be correctly termed glassware. As plastic
to Fahrenheit temperature by 9; divide the material was refined and made available to manu-
answer by 5, then add 32) facturers, plastic has been increasingly used to make
Fahrenheit to Celsius (°F − 32)5/9 (subtract 32 and laboratory supplies. A brief summary of the types
(Centigrade) divide the answer by 9; then and uses of glass and plastic commonly seen today
multiply that answer by 5) in laboratories can be found in the Navigate 2 digital
© Jones & Bartlett Learning. component. Regardless of design, most laboratory
supplies must satisfy certain tolerances of accuracy
12 Chapter 1 Basic Principles and Practices of Clinical Chemistry

and fall into two classes of precision tolerance, either rinses with appropriate grade water is recommended.
Class A or Class B as given by ASTM.12,13 Those that Check the pH of the final rinse water and compare it
satisfy Class A ASTM precision criteria are stamped with the initial pH of the prerinse water. Detergent-­
with the letter “A” on the glassware and are pre- contaminated water will have a more alkaline pH as
ferred for laboratory applications. Class B glassware compared with the pH of the prerinse water. Visual
generally have twice the tolerance limits of Class A, inspection should reveal spotless vessel walls. Any
even if they appear identical, and are often found biologically contaminated labware should be dis-
in student laboratories where durability is needed. posed of according to the precautions followed by
Vessels holding or transferring liquid are designed the laboratory.
either to contain (TC) or to deliver (TD) a specified Some determinations, such as those used in
volume. The major difference is that TC devices assessing heavy metals or assays associated with
do not deliver the volume measured when the liq- molecular testing, require scrupulously clean or dis-
uid is transferred into a container, whereas the TD posable glassware. Other applications may require
designation means that the labware will deliver the plastic rather than glass because glass can absorb
amount measured. metal ions. It is suggested that disposable glass and
Glassware used in the clinical laboratory usually plastic be used whenever possible.
fall into one of the following categories: Kimax/Pyrex Dirty reusable pipettes should be placed, with
(borosilicate), Corex (aluminosilicate), high silica, the pipette tips up, immediately in a specific pipette
Vycor (acid and alkali resistant), low actinic (amber soaking/washing/drying container. This container
colored), or flint (soda lime) glass used for dispos- should have soapy water high enough to cover
able material.14 Glassware routinely used in clinical the entire pipette. For each final water rinse, fresh
chemistry should consist of high thermal borosilicate reagent-grade water should be used; if possible,
or aluminosilicate glass. The manufacturer is the best designate a pipette container for final rinses only.
source of information about specific uses, limitations, Cleaning brushes are available to fit almost any size
and accuracy specifications for glassware. ­glassware and are recommended for any articles that
Plasticware is beginning to replace glassware are washed routinely.
in the laboratory setting; high resistance to corro- Although plastic material is often easier to clean
sion and breakage, as well as varying flexibility, has because of its nonwettable surface, it may not be
made plasticware appealing. Relatively inexpensive, it appropriate for some applications involving organic
allows most items to be completely disposable after solvents or autoclaving. Brushes or harsh abra-
each use. The major types of resins frequently used sive cleaners should not be used on plasticware.
in the clinical chemistry laboratory are polystyrene, Many initial cleaning procedures, described in
polyethylene, polypropylene, Tygon, Teflon, polycar- Appendix J (found in the Navigate 2 digital com-
bonate, and polyvinyl chloride. Again, the individual ponent), can be adapted for plasticware. Ultrasonic
manufacturer is the best source of information con- cleaners can help remove debris coating the sur-
cerning the proper use and limitations of any plastic faces of glass or p
­ lasticware. Properly cleaned lab-
material. oratory glass and plasticware should be completely
In most laboratories, glass or plastic that is in dried before using.
direct contact with biohazardous material is usu-
ally disposable. If not, it must be decontaminated
according to appropriate protocols. Should the need Laboratory Glassware
arise, cleaning of glass or plastic may require special Flasks, beakers, and graduated cylinders are used to
techniques. Immediately rinsing glass or plastic sup- hold solutions. Volumetric and Erlenmeyer flasks are
plies after use, followed by washing with a detergent two types of containers in general use in the clinical
designed for cleaning laboratory supplies and several laboratory.
distilled water rinses, may be sufficient. Presoaking A volumetric flask is calibrated to hold one exact
glassware in soapy water is highly recommended volume of liquid (TC). The flask has a round, lower
whenever immediate cleaning is impractical. Many portion with a flat bottom and a long, thin neck with
laboratories use automatic dishwashers and dry- an etched calibration line. Volumetric flasks are used
ers for cleaning. Detergents and temperature levels to bring a given reagent to its final volume with the
should be compatible with the material and the man- recommended diluent. When bringing the bottom of
ufacturer’s recommendations. To ensure that all deter- the meniscus to the calibration mark, a pipette should
gent has been removed from the labware, m ­ ultiple be used for adding the final drops of diluent to ensure
 Laboratory Equipment 13

maximum control is maintained and the calibration Table 1.4 Pipette Classification
line is not missed.
Erlenmeyer flasks and Griffin beakers are I. Design
designed to hold different volumes rather than one A. To contain (TC)
exact amount. Because Erlenmeyer flasks and Griffin B. To deliver (TD)
beakers are often used in reagent preparation, flask II. Drainage characteristics
size, chemical inertness, and thermal stability should A. Blowout
be considered. The Erlenmeyer flask has a wide bot- B. Self-draining
tom that gradually evolves into a smaller, short neck.
III. Type
The Griffin beaker has a flat bottom, straight sides,
A. Measuring or graduated
and an opening as wide as the flat base, with a small
spout in the lip. 1. Serologic
2. Mohr
Graduated cylinders are long, cylindrical tubes
3. Bacteriologic
usually held upright by an octagonal or circular base. 4. Ball, Kolmer, or Kahn
The cylinder has horizontal calibration marks and is 5. Micropipette
used to measure volumes of liquids. Graduated cylin- B. Transfer
ders do not have the accuracy of volumetric labware.
1. Volumetric
The sizes routinely used are 10, 25, 50, 100, 500, 2. Ostwald-Folin
1000, and 2000 mL. 3. Pasteur pipettes
All laboratory glassware used for critical mea- 4. Automatic macropipettes or micropipettes
surements should be Class A whenever possible to
© Jones & Bartlett Learning.
maximize accuracy and precision and thus decrease
calibration time (Figure 1.1 illustrates representative
laboratory glassware).
­ articular volume of liquid. The major difference is
p
Pipettes the amount of liquid needed to wet the interior surface
Pipettes are a type of laboratory equipment used to of the pipette and the amount of any residual liquid
transfer liquids; they may be reusable or disposable. left in the pipette tip. Most manufacturers stamp TC
Although pipettes may transfer any volume, they are or TD near the top of the pipette to alert the user as to
usually used for volumes of 20 mL or less; larger vol- the type of pipette. Like other TC-designated labware,
umes are usually transferred or dispensed using auto- a TC pipette holds or contains a particular volume but
mated pipetting devices. Table 1.4 outlines the pipette does not dispense that exact volume, whereas a TD
classification. pipette will dispense the volume indicated.
Similar to other laboratory equipment, pipettes When using either pipette, the tip must be
are designed to contain (TC) or to deliver (TD) a 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 (Figure 1.2). 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 gradua-
tion line (Figure 1.3A), and suction is then stopped.
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 liq-
uid. The liquid is allowed to drain until the bottom
of the meniscus touches the desired calibration mark
(­Figure 1.3B). 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
Figure 1.1 Laboratory glassware. the vessel (e.g., test tube, cuvette, or flask). A blowout
© Wolters Kluwer. pipette has a continuous etched ring or two small,
14 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Serologic/Mohr

Meniscus
10 Graduation line

9
A

Bottom of
Graduation line 10 the meniscus

9
B
Correct Figure 1.3 Pipetting technique. (A) Meniscus is brought
above the desired graduation line. (B) Liquid is allowed
to drain until the bottom of the meniscus touches the
Volumetric/Ostwald-Folin desired calibration mark.
© Wolters Kluwer.

measure 5, 4, 3, 2, or 1 mL of liquid, with further

graduations between each milliliter. The pipette is
designated as 5 in 1/10 increments (Figure 1.4) and
could deliver any volume in tenths of a milliliter, up
to 5 mL. Another pipette, such as a 1-mL pipette,
may be designed to dispense 1 mL and have subdivi-
sions of hundredths of a milliliter. The subgroups of
measuring or graduated pipettes are Mohr, serologic,
Correct and micropipettes. A Mohr pipette does not have
graduations to the tip. It is a self-­draining pipette,
Figure 1.2 Correct and incorrect pipette positions.
but the tip should not be allowed to touch the ves-
© Wolters Kluwer.
sel while the pipette is draining. A serologic pipette
has graduation marks to the tip and is generally a
close, ­continuous rings located near the top of the blowout pipette. A micropipette is a pipette with a
pipette. This means that the last drop of liquid should total holding volume of less than 1 mL; it may be
be expelled into the receiving vessel. Without these designed as either a Mohr or a serologic pipette.
markings, a pipette is self-draining, and the user allows Transfer pipettes are designed to dispense one
the contents of the pipette to drain by gravity. The tip volume without further subdivisions. Ostwald-Folin
of the pipette should not be in contact with the accu- pipettes are used with biologic fluids having a viscosity
mulating fluid in the receiving vessel during drainage. greater than that of water. They are blowout pipettes,
With the exception of the Mohr pipette, the tip should indicated by two etched, continuous rings at the top.
remain in contact with the side of the vessel for sev- The volumetric pipette is designed to dispense or
eral seconds after the liquid has drained. The pipette transfer aqueous solutions and is always self-draining.
is then removed (Figure 1.2). The bulb-like enlargement in the pipette stem easily
Measuring or graduated pipettes are capable
of dispensing several different volumes. Measur-
ing pipettes are used to transfer reagents or make
5 in 1/10
dilutions and can be used to repeatedly transfer a
{

particular solution. The markings at the top of a

measuring or graduated pipette indicate the vol-
ume(s) it is designed to dispense. Because the grad-
Total volume Major divisions
uation lines located on the pipette may vary, the
increments will be i­ndicated on the top of each Figure 1.4 Volume indication of a pipette.
pipette. For example, a 5-mL pipette can be used to © Wolters Kluwer.
 Laboratory Equipment 15

identifies the volumetric pipette. This type of pipette

usually has the greatest degree of accuracy and preci-
sion and should be used when diluting standards, cal-
ibrators, 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 with-
out consideration of a specific volume. These pipettes
should not be used in any quantitative analytic tech-
niques (Figure 1.5).
The automatic pipette is the most routinely
used pipette in today’s clinical chemistry labora-
tory. Automatic pipettes come in a variety of types
Figure 1.5 Disposable transfer pipettes.
including fixed volume, variable volume, and
multichannel. The term automatic, as used here, © Wolters Kluwer.

implies that the mechanism that draws up and dis-

penses the liquid is an integral part of the pipette. ­disposable. ­Figure 1.6 illustrates many common
It may be a fully ­automated/self-operating, semiau- automatic pipettes. A pipette associated with only
tomatic, or completely manually operated device. one volume is termed a fixed volume, and models
Automatic and semiautomatic pipettes have many able to select different volumes are termed vari-
advantages, including safety, stability, ease of use, able; however, only one volume may be used at a
increased precision, the ability to save time, and time. The available range of pipette volumes is
less cleaning required because the pipette tips are 1 μL to 5000 mL. A pipette with a capability of

A B

C
D
Figure 1.6 (A) Adjustable volume pipette. (B) Fixed volume pipette with disposable tips. (C) Multichannel pipette.
(D) Multichannel pipette in use.
© Wolters Kluwer.
16 Chapter 1 Basic Principles and Practices of Clinical Chemistry

less than 1 mL is ­considered a micropipette, and a as non–Class A materials, do need recalibration.15,16

pipette that dispenses greater than 1 mL is called Calibration of pipettes is done to verify accuracy
an automatic macropipette. Multichannel pipettes are and precision of the device and may be required
able to attach multiple pipette tips to a single handle by the laboratory’s accrediting agency. A gravimet-
and can then be used to dispense a fixed volume ric method (see the Navigate 2 digital component
of fluid to multiple wells, such as to a multiwell resources for this procedure) can accomplish this
microtiter plate. In addition to classification by vol- task by delivering and weighing a solution of known
ume delivery amounts, automatic pipettes can also specific gravity, such as water. A currently calibrated
be categorized according to their mechanism: air-­ analytic balance and at least Class 2 weights should
displacement, positive displacement, and dispenser be used.17 Deviation from the chosen volume is cal-
pipettes. An air-displacement pipette relies on a piston culated based on the type of pipette tested. Pipettes
for creating suction to draw the sample into a dis- that fall outside of the maximum allowable error
posable tip that must be changed after each use. The will need to be adjusted following the manufactur-
piston does not come in contact with the liquid. A er’s instructions. Although gravimetric validation
positive-displacement pipette operates by moving the is the most desirable method,18,19 pipette calibra-
piston in the pipette tip or barrel, much like a hypo- tion may also be accomplished by using photomet-
dermic syringe. It does not require a different tip ric methods, particularly for automatic pipetting
for each use. Because of carryover concerns, rinsing devices. When a spectrophotometer is used, the
and blotting between samples may be required. Dis- molar absorptivity of a compound, such as potas-
pensers and dilutor/dispensers are automatic pipettes sium dichromate, is obtained. After an aliquot of
that obtain the liquid from a common reservoir and diluent is pipetted, the change in concentration
dispense it repeatedly. The dispensing pipettes may will reflect the volume of the pipette. Another pho-
be bottle-top, motorized, handheld, or attached tometric technique used to assess pipette accuracy
to a dilutor. The dilutor often combines sampling compares the absorbances of dilutions of potassium
and dispensing functions. Many automated pipettes dichromate, or another colored liquid with appro-
use a wash between samples to eliminate carryover priate absorbance spectra, using Class A volumetric
problems. However, to minimize carryover con- labware versus equivalent dilutions made with the
tamination with manual or semiautomatic pipettes, pipetting device.
careful wiping of the tip may remove any liquid that These calibration techniques are time consum-
adhered to the outside of the tip before dispensing ing and impractical for use in daily checks. It is rec-
any liquid. Care should be taken to ensure that the ommended that pipettes be checked initially and
orifice of the pipette tip is not blotted, drawing sam- subsequently three or four times per year, or as dic-
ple from the tip. Another precaution in using manu- tated by the laboratory’s accrediting agency. Many
ally operated semiautomatic pipettes is to move the companies offer calibration services; the one chosen
plunger in a continuous and steady manner. Pipettes should also satisfy any accreditation requirements.
should be operated according to the manufacturer’s A quick, daily check for many larger volume auto-
directions. matic pipetting devices involves the use of volumet-
Disposable, one-use pipette tips are designed for ric flasks. For example, a bottle-top dispenser that
use with air-displacement pipettes. The laboratorian routinely delivers 2.5 mL of reagent may be checked
should ensure that the pipette tip is seated snugly by dispensing four aliquots of the reagent into a
onto the end of the pipette and free from any defor- 10-mL Class A volumetric flask. The bottom of the
mity. Plastic tips used on air-displacement pipettes meniscus should meet with the calibration line on
can vary. Different brands can be used for one par- the volumetric flask.
ticular pipette, but they do not necessarily perform
in an identical manner. Tips for positive-displacement
pipettes are made of straight columns of glass or plas- Syringes
tic. These tips must fit snugly to avoid carryover and Syringes are sometimes used for transfer of small vol-
can be used repeatedly without being changed after umes (< 500 μL) in blood gas analysis or in separation
each use. As previously mentioned, these devices may techniques such as chromatography or electrophore-
need to be rinsed and dried between samples to min- sis (Figure 1.7). The syringes are glass and have fine
imize carryover. barrels. The plunger is often made of a fine piece of
Class A pipettes do not need to be recalibrated by wire. Tips are not used when syringes are used for
the laboratory. Automatic pipetting devices, as well injection of sample into a gas chromatographic or
 Laboratory Equipment 17

Figure 1.7 Microliter glass syringe.

© Wolters Kluwer.

high-pressure liquid chromatographic system. In

electrophoresis work, however, disposable Teflon tips
may be used.

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
Figure 1.8 Analytic balance.
water of crystallization is removed from the com-
pound, it is said to be anhydrous. Substances that © Wolters Kluwer.

take up water on exposure to atmospheric condi-

tions are called hygroscopic. Materials that are very
­balances (Figure 1.8) are required for the preparation
hygroscopic can remove moisture from the air as well
of any primary standard. It has a single pan enclosed
as from other materials. These materials make excel-
by sliding transparent doors, which minimize envi-
lent drying substances and are sometimes used as
ronmental influences on pan movement, tared weigh-
desiccants (drying agents) to keep other chemicals
ing vessel, and sample. An optical scale allows the
from becoming hydrated. Closed and sealed contain-
operator to visualize the mass of the substance. The
ers that include desiccant material are referred to as
weight range for many analytic balances is from 0.01
desiccators and may be used to store more hygro-
mg to 160 g.
scopic substances. Many sealed packets or shipping
Electronic balances (Figure 1.9) are single-pan
containers, often those that require refrigeration,
balances that use an electromagnetic force to coun-
include some type of small packet of desiccant mate-
terbalance the weighed sample’s mass. Their measure-
rial to prolong storage.
ments equal the accuracy and precision of any avail-
able mechanical balance, with the advantage of a fast
Balances response time (< 10 seconds).
A properly operating balance is essential in produc- Test weights used for calibrating balances
ing high-quality reagents and standards. However, should be selected from the appropriate ANSI/ASTM
because many laboratories discontinued in-house Classes 1 through 4.19 Weighing instruments will
reagent preparation, balances may no longer be as need to be calibrated and adjusted periodically due
widely used. Balances are classified according to to wear and tear from frequent use. Mechanisms for
their design, number of pans (single or double), and automatic adjustments are built into many newer
whether they are mechanical or electronic or classified instruments. These instruments will test and adjust
by operating ranges. the sensitivity of the device. Periodic verification
Analytic and electronic balances are currently is still necessary to assure the performance of that
the most popular in the clinical laboratory. Analytic device. The frequency of calibration is dictated by the
18 Chapter 1 Basic Principles and Practices of Clinical Chemistry

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. Centrifuge
classification is based on several criteria, including
benchtop (Figure 1.10A) or floor model; refrigeration;
rotor head (e.g., fixed angle, hematocrit, cytocentri-
fuge, swinging bucket [Figure 1.10B], or angled); or
maximum speed attainable (i.e., ultracentrifuge).
Centrifuge maintenance includes daily cleaning
of any spills or debris, such as blood or glass, and

Figure 1.9 Electronic top-loading balance.

© Wolters Kluwer.

a­ ccreditation/licensing guidelines for a specific labo-

ratory. Balances should be kept clean and be located
in an area away from heavy traffic, large pieces of elec-
trical equipment, and open windows to prevent inac-
curate readings. The level checkpoint should always
be corrected before weighing occurs.

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 A
analysis. The centrifuge separates the mixture based
on mass and density of the component parts. It con-
sists of a head or rotor, carriers, or shields that are
attached to the vertical shaft of a motor or air com-
pressor and enclosed in a metal covering. The cen-
trifuge 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.
Centrifugal force depends on three variables:
mass, speed, and radius. The speed is expressed in rev-
olutions per minute (rpm), and the centrifugal force
generated is expressed in terms of relative centrifugal B
force (RCF) or gravities (g). The speed of the centri-
Figure 1.10 (A) Benchtop centrifuge. (B) Swinging-
fuge is related to the RCF by the following equation:
bucket rotor.
–5 2
RCF = 1 .118 × 10 × r × (rpm) © Wolters Kluwer.
 Laboratory Equipment 19

Figure 1.12 Properly loaded centrifuge.

© Wolters Kluwer.

of even placement and “opposition” (Figure 1.12).

Exact positioning of tubes depends on the design of
Figure 1.11 Properly balanced centrifuge. Colored
the centrifuge holders.
circles represent counterbalanced positions for sample
tubes. The centrifuge cover should remain closed until
the centrifuge has come to a complete stop to avoid
© Wolters Kluwer.
any aerosol production. It is recommended that
the timer, brushes (if present), and speed be peri-
ensuring that the centrifuge is properly balanced odically checked. The brushes, which are graphite
and free from any excessive vibrations. Balancing the bars attached to a retainer spring, create an electri-
centrifuge load is critical (Figure 1.11). Many newer cal contact in the motor. The specific manufacturer’s
­centrifuges will automatically decrease their speed if service manual should be consulted for details on
the load is not evenly distributed, but more often, the how to change brushes and on lubrication require-
centrifuge will shake and vibrate or make more noise ments. The speed of a centrifuge is easily checked
than expected. A centrifuge needs to be balanced using a tachometer or strobe light. The hole located
by equalizing both the volume and weight distribu- in the lid of many centrifuges is designed for speed
tion across the centrifuge head. Many laboratories verification using these devices but may also repre-
will have “balance” tubes of routinely used volumes sent an aerosol biohazard if the hole is uncovered.
and tube sizes, which can be used to match those Accreditation agencies require periodic verification
from patient samples. A good rule of thumb is one of centrifuge speeds.

CASE STUDY 1.2, PART 2

Recall Mía, the new graduate.
1. How should Mía place the chemistry tubes in the centrifuge?
2. If the centrifuge starts vibrating, what is the first troubleshooting step Mía should take?
3. If the rubber cap came off the tube during centrifugation, how should Mía decontaminate
the centrifuge?

© Ariel Skelley/DigitalVision/Getty Images.

20 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Laboratory Mathematics calculator to become acquainted with the proper use

of these functions.
and Calculations
Significant Figures pH (Negative Logarithms)
Significant figures are the minimum number of dig- In certain circumstances, the laboratorian may work
its needed to express a particular value in scientific with negative logs. Such is the case with pH or pKa.
notation without loss of accuracy. There are several As previously stated, the pH of a solution is defined
rules in regard to identifying significant figures: as the negative log of the hydrogen ion concentra-
tion. The following is a convenient formula to deter-
1. All nonzero numbers are significant (1, 2, 3, 4, 5,
mine the negative logarithm when working with pH
6, 7, 8, 9).
or pKa:
2. All zeros between nonzero numbers are
­significant. pH
3. All zeros to the right of the decimal are not signif- = x – log N(Eq. 1.11)
pK a
icant when followed by a nonzero number.
4. All zeros to the left of the decimal are not where x is the negative exponent base 10 expressed
­significant. and N is the decimal portion of the scientific notation
expression.
The number 814.2 has four significant figures, because
For example, if the hydrogen ion concentration
in scientific notation, it is written as 8.142 × 102. of a solution is 5.4 × 10−6, then x = 6 and N = 5.4.
The number 0.000641 has three significant fig- Substitute this information into Equation 1.11, and
ures, because the scientific notation expression for it becomes
this value is 6.41 × 10−4. The zeros to the right
of the decimal preceding the nonzero digits are pH = 6 – log 5.4  (Eq. 1.12)
merely holding decimal places and are not needed
to properly express the number in scientific nota- The logarithm of N (5.4) is equal to 0.7324, or 0.73.
tion. However, by convention, zeros following a The pH becomes
decimal point are considered significant. For exam- pH = 6 – 0.73 = 5.27 (Eq. 1.13)
ple, 10.00 has four significant figures. The zeros to
the right of the decimal indicate the precision of The same formula can be applied to obtain the
this value. hydrogen ion concentration of a solution when only
the pH is given. Using a pH of 5.27, the equation
becomes
Logarithms
Logarithms are the inverse of exponential functions 5.27 = x – log N (Eq. 1.14)
and can be related as such:
In this instance, the x term is always the next larg-
x = A or B = logA (x)
B est whole number. For this example, the next largest
whole number is 6. Substituting for x, the equation
This is then read as B is the log base A of X, where becomes
B must be a positive number, A is a positive num-
ber, and A cannot be equal to 1. Calculators with a 5.27 = 6 – log N(Eq. 1.15)
log function do not require conversion to scientific A shortcut is to simply subtract the pH from
notation. x (6 − 5.27 = 0.73) and take the antilog of that
To determine the original number from a log answer 5.73. The final answer is 5.73 × 10−6. Note
value, the process is performed in reverse. This pro- that rounding, while allowed, can alter the answer.
cess is termed the antilogarithm or antilog as it is the A more algebraically correct approach follows in
inverse of the logarithm. Most calculators require Equations 1.16 through 1.18. Multiply all the vari-
using an inverse or secondary/shift function when ables by −1:
entering this value. If given a log of 3.1525, the
resulting value is 1.424 × 103 on the base 10 system. ( –1)(5 .27) = (–1)(6) – (–1)(log N )
(Eq. 1.16)
Consult the specific manufacturer’s directions of the –5.27 = –6 + log N
 Laboratory Mathematics and Calculations 21

Solve the equation for the unknown quantity by add- Example 1.2: Weight/Volume (w/v)
ing a positive 6 to both sides of the equal sign, and the
equation becomes The most frequently used term for a percent solu-
tion is weight per volume, which is often expressed
6 – 5.27 = log N as grams per 100 mL of the diluent. To make up
(Eq. 1.17)
0.73 = log N 1000 mL of a 10% (w/v) solution of NaOH, use the
preceding approach. Restate the w/v as a fraction:
The result is 0.73, which is the antilogarithm value of
N, which is 5.37, or 5.4: 10 g
10% = = 0.10
100 mL
Antilog 0.73 = N; N = 5.37 = 5.4 (Eq. 1.18)

The hydrogen ion concentration for a solution with a Next, set up a ratio and solve for x
pH of 5.27 is 5.4 × 10−6. Many scientific calculators 10g x
have an inverse function that allows for more direct =
100 mL 1000 mL (Eq. 1.22)
calculation of negative logarithms.
x = 100g
Concentration Lastly, add 100 g of 10% NaOH to a 1000-mL vol-
A description of each concentration term is pro- umetric Class A flask and add sufficient volume of
vided at the beginning of this chapter. The follow- reagent-grade water to the calibration mark.
ing discussion focuses on the basic mathematical
expressions needed to prepare reagents of a stated Example 1.3: Volume/Volume (v/v)
concentration.
If both chemicals in a solution are in the liquid form,
the volume per unit volume is used to give the vol-
Percent Solution ume of solute present in 100 mL of solution. To make
A percent solution is determined in the same man- up 50 mL of a 2% (v/v) concentrated hydrochloric
ner regardless of whether weight/weight, volume/ acid solution, a similar approach is used. The (v/v) is
volume, or weight/volume units are used. Percent restated as a fraction:
implies “parts per 100,” which is represented as per-
cent (%) and is independent of the molecular weight 2 mL
2% = = 0.02
of a substance. 100 mL

Example 1.1: Weight/Weight (w/w) Then, the calculation becomes

To make up 250 g of a 5% aqueous solution of hydro- 0.02 × 50 mL = 1 mL

chloric acid (using 12 M HCl), multiply the total
amount by the percent expressed as a decimal. The or using a ratio
5% aqueous solution can be expressed as 2 mL x
=
5 100 mL 50 mL(Eq. 1.23)
5% = = 0.050(Eq. 1.19)
100 x = 1 mL
Therefore, the calculation becomes Therefore, add 40 mL of reagent-grade water to a
50-mL Class A volumetric flask, add 1 mL of concen-
0.050 × 250 g = 12.5 g of 12M HCl (Eq. 1.20)
trated HCl, mix, and dilute up to the calibration mark
Another way of arriving at the answer is to set up a with reagent-grade water. Remember, always add acid
ratio so that to water!

Desired solution concentration = Final

Molarity
product of 12 M HCl
Molarity (M) is routinely expressed in units of moles
5 x per liter (mol/L) or sometimes millimoles per millili-
=
100 250 (Eq. 1.21) ter (mmol/mL). Remember that 1 mol of a substance
x =12.5 is equal to the gram molecular weight (gmw) of that
22 Chapter 1 Basic Principles and Practices of Clinical Chemistry

substance. When trying to determine the amount of 40 g/mol. Rearrange the equation so that
substance needed to yield a particular concentration, grams can be canceled, and the remaining
initially decide what final concentration units are units reflect those needed in the answer,
needed. For molarity, the final units will be moles per which are mole/L.
liter (mol/L) or millimoles per milliliter (mmol/mL). Step 3: The equation becomes
The second step is to consider the existing units and
the relationship they have to the final desired units. 24 g NaOH 1mol mol
× = 0.6 (Eq. 1.25)
Essentially, try to put as many units as possible into L 40 g NaOH L
like terms and arrange so that the same units cancel
each other out, leaving only those needed in the final By canceling out like units and performing the appro-
answer. To accomplish this, it is important to remem- priate calculations, the final answer of 0.6 M or
ber what units are used to define each concentration 0.6 mol/L.
term. It is key to understand the relationship between
molarity (moles/liter), moles, and gmw. While molar- Example 1.6
ity is given in these examples, the approach for molal-
Prepare 250 mL of a 4.8 M solution of HCl.
ity is the same except that one molal is expressed as
one mole of solute per kilogram of solvent. For water, Step 1: Start with what is given: 4.8 moles/L
one kilogram is proportional to one liter, so molarity Step 2: Determine the gmw of HCl ([H = 1] +
and molality are equivalent. [Cl = 35.5] = 36.5 g/mol)

Example 1.4 Step 3: Set up the equation, cancel out like

units, and perform the appropriate calculations:
How many grams are needed to make 1 L of a 2 M
4.8 mol 36.5 g HCI 1L
solution of HCl? × ×
1L 1 mol 1000 mL(Eq. 1.26)
Step 1: What units are needed in the final
answer? Answer: Grams per liter (g/L). × 250 mL = 43.8 g HCI
Step 2: Assess other mass/volume terms In a 250-mL Class A volumetric flask, add 200 mL
used in the problem. In this case, moles are of reagent-grade water. Add 43.8 g of HCl and mix.
also needed for the calculation: How many Dilute up to the calibration mark with reagent-grade
grams are equal to 1 mole? The gmw of HCl, water.
which can be determined from the periodic
table, will be equal to 1 mole. For HCl, the
gmw is 36.5g/mol, so the equation may be Normality
written as Normality (N) is expressed as the number of equiv-
alent weights per liter (Eq/L) or milliequivalents per
36.5 g HCl 2 mol 73 g HCl
 = (Eq. 1.24) milliliter (mmol/mL). Equivalent weight is equal
mol L L to gmw divided by the valence (V). Normality has
Cancel out like units, and the final units should be often been used in acid–base calculations because an
grams per liter. In this example, 73 g HCl per liter is equivalent weight of a substance is also equal to its
needed to make a 2 M solution of HCl. combining weight (or the weight that will combine
with or displace 1 mole of hydrogen). Another advan-
Example 1.5 tage in using equivalent weight is that an equivalent
weight of one substance is equal to the equivalent
A solution of NaOH is contained within a Class A weight of any other chemical.
1-L volumetric flask filled to the calibration mark.
The content label reads 24 g of NaOH. Determine the Example 1.7
molarity.
Give the equivalent weight, in grams, for each sub-
Step 1: What units are needed? Answer: Moles stance listed below.
per liter (mol/L).
1. NaCl (gmw = 58 g/mol, valence = 1)
Step 2: The units that exist are grams and
L. NaOH may be expressed as moles and 58 g
= 58 g per equivalent weight(Eq. 1.27)
grams. The calculated gmw of NaOH is L
 Laboratory Mathematics and Calculations 23

2. H2SO4 (gmw = 98 g/mol, valence = 2) e­xisting units are exchanged for units needed. The
equation is
98 g
= 49 g per equivalent weight(Eq. 1.28) 2.5 Eq HCl 36.5 g × HCl 1 mol HCl
2 × ×
L 1 Eq 36.5 g HCl (Eq. 1.33)
Example 1.8 = 2.5 mol/L HCl
What is the normality of a 500 mL solution that con- The second approach is to use the normality-to-­
tains 7 g of H2SO4? molarity conversion formula. The equation now
Step 1: What units are needed? Answer: becomes
Normality expressed as equivalents per liter M × V = 2.5 N
(Eq/L).
V =1
Step 2: Start with what is given: 7 g/500 mL  (Eq. 1.34)
2.5 N
M= = 2.5 N
Step 3: Calculate the gmw of H2SO4 (98 g/mol) 1
and determine the valence (2)
When the valence of a substance is 1, the molar-
Step 4: Add a conversion factor to convert mL ity will equal the normality. As previously men-
to L (1000 mL/1 L) tioned, normality either equals or is greater than the
Step 5: Cancel out like terms and calculate molarity.
the result in Eq/L. Although there are various methods to calcu-
late laboratory mathematical problems, the tech-
This equation is:
nique of using conversion factors and canceling like
7 g H2SO4 1 Eq 1000 mL units is used in most clinical chemistry calculation,
× × regardless of whether the problem requests molar-
500 mL 49 g H2SO4 1 L (Eq. 1.29)
ity and normality or exchanging one concentration
= 0.285 Eq/L = 0.285 N term for another. However, it is necessary to recall
the interrelationship between all the units in the
Example 1.9 expression.
What is the normality of a 0.5 M solution of H2SO4?
Specific Gravity
Continuing with the previous approach, the final
equation is Density is expressed as mass per unit volume of a
substance. The specific gravity is the ratio of the
0.5 mol H2SO4 98 g H2SO4 1 EqH2SO4 density of a material when compared with the den-
× × sity of pure water at a given temperature and allows
L mol H2SO4 49 g H2SO4
the laboratorian a means of expressing density in
= 1 Eq/L = 1N terms of volume. The units for density are grams per
(Eq. 1.30) milliliter (g/mL). Specific gravity is often used with
When converting between molarity and normal- very concentrated materials, such as commercial
ity, the following conversion formula may be acids (e.g., sulfuric and hydrochloric acids).
applied: The density of a concentrated acid can also be
expressed in terms of an assay or percent purity.
M × V = N (Eq. 1.31) The actual concentration is equal to the specific
­gravity multiplied by the assay or percent purity value
where V is the valence of the compound. Using this (expressed as a decimal) stated on the label of the
formula, Example 1.9 becomes container.
0.5 M × 2 = 1N (Eq. 1.32) Example 1.11
Example 1.10 What is the actual weight of a supply of concentrated
HCl on which the label reads, specific gravity 1.19
What is the molarity of a 2.5 N solution of HCl? with an assay value of 37%?
This problem may be solved in several ways.
One way is to use the stepwise approach in which 1.19 g/mL × 0.37 = 0.44g/mL of HCl (Eq. 1.35)
24 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Example 1.12 Example 1.14

What is the molarity of this same stock solution? The What volume is needed to make 500 mL of a
final units desired are moles per liter (mol/L). The 0.1 M solution of Tris buffer from a solution of 2 M
molarity of the solution is Tris ­buffer?
0.44 g HCl Identify the known values:
1 mol HCl 1000 mL
× × Concentration of initial substance (C1) = 2 M
mL 36.5 g HCl L (Eq. 1.36)
= 12.05 mol/L or 12M Volume of the product (V2) = 500 mL
Concentration of the product (C2) = 0.1 M
Conversions And the equation becomes:
To convert one unit into another, the same approach
of canceling out like units can be applied. In some V1 × 2 M = 0.1 M × 500 mL
instances, a chemistry laboratory may report a given (V1)(2 M) = (0.1 M)(500 mL);
analyte using two different concentration units— (V1)(2 M) = 50 mL
for example, calcium. The recommended SI unit
for calcium is millimoles per liter. The more tra- 50 mL
ditional units are milligrams per deciliter (mg/dL). Therefore, V1 = 25 mL (Eq. 1.39)
2
Again, it is important to understand the relationship
between the units given and those needed in the It requires 25 mL of the 2 M solution to make up
final answer. 500 mL of a 0.1 M solution.
This problem differs from the other conversions
in that it is actually a dilution of a stock solution.
Example 1.13
While this approach will provide how much stock
Convert 8.2 mg/dL calcium to millimoles per liter is needed when making the solution, the laborato-
(mmol/L). The gmw of calcium is 40 g. If there are rian must subtract the obtained volume value from
40 g per mol, then it follows that there are 40 mg per the final desired volume to determine the amount of
mmol. The final units needed are mmol/L. The equa- diluent needed, in this case 475 mL. A more involved
tion becomes discussion of dilution problems follows.
8.2 mg
10 dL 1 mmol 2.05 mmol
× × = Dilutions
dL 1L 40 mg L
A dilution represents the part(s) of concentrated
(Eq. 1.37)
material to the total final volume of a solution. It
Once again, the systematic stepwise approach of can- consists of the parts of the substance being diluted
celing similar units can be used for this conversion in the total numbers of parts of the solution. In con-
problem. trast, ratio refers to part substance to part substance.
A frequently encountered conversion prob- The most common dilution uses one part patient
lem or, more precisely, a dilution problem occurs serum plus one part saline. This is a 1:1 ratio of
when a weaker concentration or different volume serum to saline. It is a 1:2 dilution which can also be
is needed than the stock substance available, but expressed as a fraction (½ dilution). After analysis,
the concentration terms are the same. The follow- the laboratory result is multiplied by the reciprocal
ing formula is used where V1 is the volume of the of the dilution (2/1) which is known as the dilution
first substance, C1 is the concentration of the first factor. Dilutions are required when the result is above
substance, V2 is the volume of the second sub- the linearity of the assay. A dilution is an expression
stance, and C2 is the concentration of the second of concentration. Because a dilution is made by add-
substance: ing a more concentrated substance to a diluent, the
dilution is always less concentrated than the original
V1 × C1 = V2 × C2(Eq. 1.38)
substance. There is an inverse relationship between
This formula is useful only if the concentration and the dilution factor and concentration. As the dilution
volume units between the substances are the same and factor increases, the concentration decreases. A dilu-
if three of four variables are known. tion can be expressed as either a fraction or a ratio.20
 Laboratory Mathematics and Calculations 25

Example 1.15 Example 1.17

What dilution is needed to make a 100 mmol/L The formula (V1)(C1) = (V2)(C2) may also be used for
sodium solution from a 3000 mmol/L stock simple dilution calculations. This is acceptable, as
solution? long as stock volume is subtracted from the total final
volume for the correct diluent volume.
1 00 mmol L 1
× = (Eq. 1.40) (V1)(C1) = (V2)(C2)(x)(3000 mmol/L)
L 3000 mmol 30
The dilution indicates 1 part stock solution is = (150 mL)(100 mmol/L)
needed to make a total volume of 30 mL. To pre-
pare this dilution, 1 mL of stock solution is added x=5
to 29 mL of diluent to achieve a total final vol- 150 – 5 = 145 mL of diluent shouldbe added to 5 mL
ume of 30 mL. Note that the dilution indicates the of stock (Eq. 1.42)
parts per total amount. In making the dilution,
the sum of the amount of the stock material plus
the amount of the diluent must equal the total
Simple Dilutions
volume. When making a simple dilution, the laboratorian must
It is important to differentiate when a dilution decide on the total volume desired and the amount of
or a ratio is stated within a procedure. For exam- stock to be used.
ple, making a “1-in-4” dilution means adding one
Example 1.18
part stock to obtain a total of four parts. That is,
one part of stock would be added to three parts of A 1/10 dilution of serum can be achieved by using any
diluent. The dilution would be 1/4. Analyses per- of the following approaches. A ratio of 1:9—one part
formed on the diluted material would need to be serum and nine parts diluent (saline) can be achieved
multiplied by 4, the dilution factor, to get the final using the following:
concentration. The dilution factor is the reciprocal
of the dilution. This is very different from a pro- • 100 μL of serum added to 900 μL of saline
cedure indicating preparation of a “1-to-4” ratio! • 20 μL of serum added to 180 μL of saline
In this instance, the dilution would be 1/5, where • 1 mL of serum added to 9 mL of saline
there is 1 part of serum and 4 parts of diluent. It is • 2 mL of serum added to 18 mL of saline
important that you fully understand the meaning of Note that the sum of the ratio of serum to diluent
these expressions. Dilutions should be made using (1:9) needed to make up each dilution satisfies the
reagent-grade water, saline, or method-specific dilu- dilution factor (1/10) of stock material to total vol-
ent. The diluted sample should be thoroughly mixed ume. When thinking about the stock to diluent vol-
before analysis. ume, subtract the parts of stock needed from the total
volume or parts to get the number of diluent “parts”
needed. Once the volume of each part, usually stock,
Example 1.16
is known, multiply the diluent parts needed to obtain
Make 150 mL of the 100 mmol/L sodium solution the correct volume.
using a 1:30 dilution.
Begin with the dilution and set up a ratio between Example 1.19
the desired part (mL) to the total parts (150 mL) to You have a 10 g/dL stock of protein standard. You
determine the amount of sodium solution needed. need a 2 g/dL standard. You only have 0.200 mL of 10
The equation becomes g/dL stock to use. The procedure requires 0.100 mL.
1 x mL Solution:
=
30 150 mL  (Eq. 1.41) 2 g/dL 1
= = Dilution(Eq. 1.43)
x = 5 mL 10 g/dL 5
Note that 5/150 reduces to the dilution of 1/30. To You will need 1 part or volume of stock in a
make up this solution, 5 mL of sodium solution is total of 5 parts or volumes. Subtracting 1 from
added to 145 mL of diluent, making the total volume 5 yields that 4 parts or volumes of diluent is needed
equal to 150 mL. (Figure 1.13). In this instance, you need at least
26 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Dilution factor: 1/2

1 Stock “part” or volume
Dilution result = 8.6 mg/dL
2
Because this result represents 1/2 of the concen-
3
“Parts” or volumes of diluent tration, the inverse (or reciprocal) of the dilution is
4
needed to make a 1/5 dilution used, and the serum creatinine value must take into
consideration this dilution, so the actual value is
5
2 × 8.6 mg/dL = 17.2 mg/dL (Eq. 1.44)
Figure 1.13 Simple dilution. Consider this diagram
depicting a substance having a 1/5 dilution factor. The
dilution factor represents that 1 part of stock is needed
Serial Dilutions
from a total of 5 parts. To prepare this dilution, you would A serial dilution may be defined as multiple, pro-
determine the volume of 1 “part,” usually the stock or gressive dilutions that dilutes highly concentrated
patient sample. The remainder of the “parts” or total solutions to produce solutions with lower concen-
would constitute the amount of diluent needed, or four tration. Serial dilutions are extremely useful when
times the volume used for the stock. the volume of concentrate or diluent is in short sup-
© Wolters Kluwer. ply and its use needs to be minimized, or when a
number of dilutions are required, such as in deter-
0.100 mL for the procedure. You have 0.200 mL of mining a titer. For instance, the volume of patient
stock. You can make the dilution in various ways, as sample available to the laboratory may be small
seen in Example 1.20. (e.g., pediatric samples), and a serial dilution may
be needed to ensure that sufficient sample is avail-
Example 1.20 able for analysis.
The serial dilution is initially made in the same
There are several ways to prepare a 1/5 dilution hav- manner as a simple dilution. Subsequent dilutions will
ing only 0.200 mL of stock and needing a total min- then be made from each preceding dilution. When a
imum volume of 0.100 mL. serial dilution is made, certain criteria may need to
be satisfied. The criteria vary with each situation but
• Add 0.050 mL stock (1 part) to 0.200 mL of dilu-
usually include such considerations as the total vol-
ent (4 parts × 0.050 mL).
ume desired, the amount of diluent or concentrate
• Add 0.100 mL of stock (1 part) to 0.400 mL of
available, the dilution factor, the final concentration
diluent (4 parts × 0.100 mL).
needed, and the support materials required.
• Add 0.200 mL of stock (1 part) to 0.800 mL of
diluent (4 parts × 0.200 mL).
Example 1.23
The dilution factor is also used to determine the
final concentration of a dilution by multiplying the A three-tube, twofold serial dilution is to be made on
original concentration by the inverse of the dilution a sample. To start, the three tubes must be labeled. It
factor or the dilution factor denominator when it is is arbitrarily decided that the total volume for each
expressed as a fraction. dilution is to be 1 mL. Into the first tube, 0.5 mL
of diluent is added and then 0.5 mL of patient sam-
Example 1.21 ple. This satisfies the “twofold” or 1/2 dilution for
tube 1. In the next tube, 0.5 mL of diluent is again
Determine the concentration of a 200 μmol/mL added, along with 0.5 mL of well-mixed liquid from
human chorionic gonadotropin (hCG) standard that tube 1. This satisfies the 1/2 dilution in tube 2,
was diluted 1/50. This value is obtained by multiply- bringing the total tube dilution to 1/4. For the third
ing the original concentration, 200 μmol/mL hCG, tube, 0.5 mL of diluent is added, along with 0.5 mL
by the dilution factor, 1/50. The result is 4 μmol/mL of well-mixed liquid from tube 2. This satisfies the
hCG. Quite often, the concentration of the original 1/2 dilution within the tube but brings the total tube
material is needed. dilution to 1/8. The calculation for these values is
Example 1.22 1 1
(Tube 1 Dilution) × (Tube 2 Dilution)
A 1/2 dilution of serum with saline had a creatinine 2 2
(Eq. 1.45)
result of 8.6 mg/dL. Calculate the actual serum creat- 1
= total dilution for tube 2
inine concentration. 4
 Laboratory Mathematics and Calculations 27

1 mL 1 mL 1/20 × (x) = 1/100

1/2 1/4
mixture- mixture where (x) = 5 (or 1 part stock to 4 parts diluent)
#1 primary #2 #3
dilution 1/100 × (x) = 1/200
1/2 1/4 1/8
where (x) = 2 (or 1 part stock to 1 part diluent)
(Eq. 1.47)
} 1 mL serum } 1dilution
mL primary
} 11/4mLmixture In practice, the 1/10 dilution must be diluted by
1 mL diluent 1 mL diluent 1 mL diluent
a factor of 2 to obtain a subsequent 1/20 dilution.
Because the second tube already contains 2 mL of
Figure 1.14 Serial dilution.
diluent, 2 mL of the 1/10 dilution should be added
© Wolters Kluwer.
(1 part stock to 1 part diluent). In preparing the
1/100 dilution from this, a 1/5 dilution factor of
the 1/20 mixture is required (1 part stock to 4 parts
Making a 1/2 dilution of the 1/4 dilution will diluent). Because this tube already contains 2 mL,
result in the next dilution (1/8) in Tube 3. To establish the volume of diluent in the tube is divided by its
the dilution factor needed for subsequent dilutions, it parts, which is 4; thus, 500 μL, or 0.500 mL, of
is helpful to solve the following equation for (x): stock should be added. The 1/200 dilution is pre-
pared in the same manner using a 1/2 dilution
Stock/preceding concentration × (x)
(Eq. 1.46) factor (1 part stock to 1 part diluent) and adding
= (final dilution factor)
2 mL of the 1/100 to the 2 mL of diluent already in
Refer to Figure 1.14 for an illustration of this serial the tube.
dilution.
Water of Hydration
Example 1.24 Some compounds are available in a hydrated form.
Another type of dilution combines several dilution A reagent protocol often designates the use of an
factors that are not multiples of one another. In our anhydrous form of a chemical; frequently, however,
previous example, 1/2, 1/4, and 1/8 dilutions are all all that is available is a hydrated form. To obtain a
related to one another by a factor of 2. Consider the correct gmw for these chemicals, the attached water
situation when 1/10, 1/20, 1/100, and 1/200 dilution molecule(s) must be included.
factors are required. There are several approaches to
solving this type of dilution problem. One method Example 1.25
is to treat the 1/10 and 1/20 dilutions as one serial How much CuSO4·5H2O must be weighed to prepare
dilution problem, the 1/20 and 1/100 dilutions as a 1 L of 0.5 M CuSO4? When calculating the gmw of
second serial dilution, and the 1/100 and 1/200 dilu- this substance, the water weight of 90 g must be con-
tions as the last serial dilution. Another approach is sidered so that the gmw is 250 g rather than gmw of
to consider what dilution factor of the concentrate CuSO4 alone (160 g). Therefore,
is needed to yield the final dilution. In this example,
the initial dilution is 1/10, with subsequent dilutions 250 g CuSO4 5H2O 0.5 mol
of 1/20, 1/100, and 1/200. The first dilution may be × = 125 g/L(Eq. 1.48)
mol 1 g
accomplished by adding 1 mL of stock to 9 mL of
diluent. The total volume of solution is 10 mL. Our Cancel out like terms to obtain the result of 125 g/L of
initial dilution factor has been satisfied. In making the the hydrated form CuSO4·5H2O.
remaining dilutions, 2 mL of diluent is added to each
test tube. Example 1.26

Initial/preceding dilution × (x) = dilution needed A procedure requires 0.9 g of CuSO4. All that is avail-
able is CuSO4·5H2O. What weight of CuSO4·5H2O
Solve for (x). is needed? Calculate the percentage of CuSO4 pres-
Using the dilution factors listed above and solving ent in CuSO4·5H2O. Again, using the gmw, the per-
for (x), the equations become centage is
1/10 × (x) = 1/20 160
= 0.64, or 64%(Eq. 1.49)
where (x) = 2 (or 1 part stock to 1 part diluent) 250
28 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Therefore, 1 g of CuSO4·5H2O contains 0.64 g of

CuSO4, so the equation becomes 0.4

Absorbance
0.9 g CuSO 4 needed 0.3
0.64 CuSO 4 in CuSO 4 ⋅ 5H 2O (Eq. 1.50) 0.2
= 1.41 g CuSO 4  5H 2O required
0.1

Graphing and Beer’s Law 1 2 3 4 5

The Beer-Lambert law (Beer’s law) mathematically Concentration
establishes the relationship between analyte concen-
tration and absorbance of light in many photometric Unknown absorbance = 0.250
Concentration from graph = 3.2
determinations. Beer’s law states that the concen-
tration of a substance is directly proportional to the Figure 1.15 Standard curve.
amount of light absorbed or inversely proportional to © Wolters Kluwer.
the logarithm of the light transmitted. Beer’s law can
be expressed as
defined as the ratio of the radiant energy transmit-
A = abc(Eq. 1.51) ted divided by the radiant incident energy of the
­sample (I). The mathematical derivation of %T can
where A is absorbance; a is the absorptivity constant
be expressed as
for a particular compound at a given wavelength
under specified conditions (such as temperature and I
%T = × 100(Eq. 1.52)
pH); b is the length of the light path; and c is the I0
­concentration.
If a method follows Beer’s law, then absorbance Where I0 is the incident light, and I is the trans-
is proportional to concentration as long as the length mitted light. The relationship between transmission
of the light path and the absorptivity of the absorb- and absorbance yields a nonlinear curve, and can be
ing species remain unaltered during the analysis. In calculated using the following formula:
practice, however, there are limits to the predictabil- A = – log ( I / I 0 ) = log (100 %) – log %T
ity of a linear response. In automated systems, adher- (Eq. 1.53)
ence to Beer’s law is often determined by checking = 2 – log%T
the linearity of the test method over a wide concen- Once a standard curve has been established, it is
tration range. The limits of linearity often represent permissible to run just one standard, or calibrator, as
the reportable range of an assay. This term should long as the system remains unchanged. A One-point
not be confused with the reference ranges associated calibration or calculation refers to the calculation
with clinical significance of a test. Assays measuring of the comparison of the known standard or calibra-
absorbance generally obtain the concentration results tor concentration and its corresponding absorbance
by using a graph of Beer’s law, known as a standard to the absorbance of an unknown value accord-
graph or curve. This graph is made by plotting absor- ing to the following ratio, where Cu and Au indicate
bance versus the concentration of known standards the concentration and absorbance, respectively, for
(Figure 1.15). Because most photometric assays set the unknown value:
the initial absorbance to zero (0) using a reagent
blank, the initial data points are 0,0. Graphs should Concentration of standard (Cs )
=
be labeled properly and the concentration units Absorbance of standard (As )
must be given. The horizontal axis is referred to as (Eq. 1.54)
Concentration of standard (Cu )
the x-axis, whereas the vertical line is the y-axis.
Absorbance of standard (Au )
By convention in the clinical laboratory, concentra-
tion is usually plotted on the x-axis. On a standard Solving for the concentration of the unknown, the
graph, only the standard and the associated absor- equation becomes
bances are plotted.
In terms of transmitted light, Beer’s law is ( Au )(Cs )
Cu = (Eq. 1.55)
expressed as percent transmission (%T), where T is As
 Laboratory Mathematics and Calculations 29

Example 1.27 enzyme that will catalyze 1 μmol of substrate per

minute per liter. These units were often expressed
The biuret assay method for protein is very stable and as units per liter (U/L). The designations IU, U, and
follows Beer’s law. Rather than make up a standard IU/L were adopted by many clinical laboratories
graph, one protein standard of 6 g/dL concentration to represent the IU. Although the reporting unit is
was assayed. The measured absorbance of the stan- the same, unless the analysis conditions are iden-
dard was 0.400, and the measured absorbance of
tical, use of the IU does not standardize the actual
the unknown was 0.350. Determine the value of the
enzyme activity, and therefore, results between dif-
unknown in g/dL.
ferent methods of the same enzyme do not result
(0.350)(6 g/dL) in equivalent activity of the enzyme. For example,
Cu = = 5.25 g/dL(Eq. 1.56)
(0.400) an alkaline phosphatase performed at 37°C will
catalyze more substrate than if it is run at lower
This method of calculation is acceptable as long temperature, such as 25°C, even though the unit
as everything in the system, including the instru- of expression, U/L, will be the same. The SI rec-
ment and lot number of reagents, remains the same. ommended unit is the katal, which is expressed as
If anything in the system changes, a new standard moles per liter per second. Whichever unit is used,
graph should be generated. Verification of linearity calculation of the activity using Beer’s law requires
and/or calibration is required whenever a system
inclusion of the dilution and, depending on the
changes or becomes unstable. Regulatory agen-
reporting unit, possible conversion to the appropri-
cies often prescribe the condition of verification
ate term (e.g., μmol to mol, mL to L, minute to sec-
as well as how frequently the linearity needs to be
ond, and temperature factors). Beer’s law for the IU
performed.
now becomes

Enzyme Calculations ( ∆A )106 ( TV)

C  (Eq. 1.59)
Another application of Beer’s law is the calculation (ε )(b)(SV)
of enzyme assay results. When calculating enzyme
where TV is the total volume of sample plus reagents
results, the rate of change in absorbance over time
in mL and SV is the sample volume used in mL. The
is often monitored continuously during the reac-
tion to give the difference in absorbance, known as 10−6 converts moles to μmol for the IU. If another
the delta absorbance, or ∆A. Instead of using a unit of activity is used, such as the katal, conver-
standard graph or a one-point calculation, the molar sion into liters and seconds would be needed,
absorptivity of the product is used. If the absorptivity but the conversions to and from micromoles are
constant and absorbance, in this case ∆A, are known, excluded.
Beer’s law can be used to calculate the enzyme con-
centration directly without the need of a standard Example 1.28
graph, as follows:
The ∆A per minute for an enzyme reaction is 0.250.
A = abC The product measured has a molar absorptivity
A (Eq. 1.57) of 12.2 × 103 at 425 nm at 30°C. The incubation
C=
ab and reaction temperature are also kept at 30°C.
The assay calls for 1 mL of reagent and 0.050 mL of
When the absorptivity constant (a) is given in sample. Give the enzyme activity results in interna-
units of grams per liter (moles) through a 1-­centimeter
tional units.
(cm) light path, the term molar absorptivity (ε) is used.
Applying Beer’s law and the necessary conver-
Substitution of ε for a and ∆A for A produces the fol-
sion information, the equation becomes
lowing Beer’s law formula:
∆A (0.250)(106 )(1.050 mL)
C = (Eq. 1.58) C= = 430 U (Eq. 1.60)
ε (12.2×103 )(1)(0.050 mL)

For reporting enzyme activity, the IU, or Note: b is usually given as 1 cm; because it is a con-
­international unit, is defined as the amount of stant, it may not be considered in the calculation.
30 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Specimen Collection In addition to venipuncture, blood samples can

be collected using a capillary puncture technique that
and Handling involves using either the outer area of the bottom of
The process of specimen collection, handling, and the foot (a heel stick) for infants or the lateral side of
processing remains one of the primary areas of pre- the middle or ring finger for individuals 1 year and
analytic errors. Careful attention to each phase of the older (finger stick). A sharp lancet device is used to
testing process is necessary to ensure proper subse- pierce the skin, and an appropriate capillary or micro-
quent testing and reporting of accurate and reliable tainer tube is used for sample collection.22
results. All accreditation agencies require laboratories Analytic testing of blood involves the use of
to clearly define and delineate the procedures used whole blood, serum, or plasma. Whole blood, as
for proper collection, transport, and processing of the name implies, contains the liquid portion of the
patient samples and the steps used to minimize and blood, called plasma, and its cellular components
detect any errors, along with the documentation of (red blood cells, white blood cells, and platelets).
the resolution of any errors. The Clinical Laboratory The collection of whole blood requires the vacuum
Improvement Amendments Act of 1988 (CLIA 88)21 tube to contain an anticoagulant. Complete mixing
specifies that procedures for specimen submission of the blood immediately following venipuncture
and proper handling be documented, including the is necessary to ensure the anticoagulant adequately
disposition of any specimen that does not meet the inhibits the specimen from clotting. As the tube of
laboratories’ criteria of acceptability. whole blood settles, the cells fall toward the bottom,
leaving a clear yellow supernatant on top, which is
the plasma.
Types of Samples If a tube does not contain an anticoagulant, the
Phlebotomy, or venipuncture, is the act of obtaining blood forms a fibrin clot incorporating the cells;
a blood sample from a vein using a needle attached this clot consumes fibrinogen. The remaining liquid
to a collection device or a stoppered evacuated tube. is called serum rather than plasma (Figure 1.16).
The collection tubes are available in different vol- Most testing in the clinical chemistry laboratory is
ume sizes: from pediatric sizes (≈150 μL) to larger performed on either plasma or serum. The major
10 mL tubes. The most frequent site for venipunc- difference between plasma and serum is that serum
ture is the medial antecubital vein of the arm. A tour- does not contain fibrinogen and some potassium
niquet made of pliable nonlatex rubber flat band or is released from platelets (i.e., potassium levels are
tubing is wrapped around the arm, causing cessa- slightly higher in serum than in plasma). It is import-
tion of blood flow and dilation of the veins, mak- ant that serum samples be allowed to completely clot
ing for easier detection. The gauge of the needle is (≈30 minutes) in an upright position at room tem-
inversely related to the size of the needle; the larger perature before being centrifuged. Plasma samples
the number, the smaller the needle bore and length. also require centrifugation but do not need time to
Routine venipuncture uses a 23- or 21-gauge needle. clot, decreasing the turnaround time for testing and
A winged infusion set, sometimes referred to as a but- reporting results.
terfly because of the appearance of the setup, may be Centrifugation of the sample accelerates the pro-
used whenever the veins are fragile, small, or difficult cess of separating the liquid portion and cellular por-
to detect. The butterfly needle is attached to a piece tion. Specimens should be centrifuged according to
of tubing, which is then attached to a hub or barrel. recommendations by the tube manufacturer or test
Because of potential for needlesticks and cost of the protocol, usually approximately 10 minutes at an RCF
product, this practice may be discouraged. However, of 1000 to 2000 g, but should avoid the mechanical
newer push-button safety devices are now available. destruction of red blood cells that can result in hemo-
When selecting an appropriate vein, sites adja- globin release, which is called hemolysis.
cent to IV (intravenous) therapy should be avoided. Arterial blood samples measure blood gases
If both arms are involved in IV therapy and the IV (partial pressures of oxygen and carbon dioxide) and
cannot be discontinued for a short time, a site below pH. Syringes containing heparin anticoagulant are
the IV site may be considered. In such cases, the used instead of evacuated tubes because of the pres-
initial sample drawn (5 mL) should be discarded sure in an arterial blood vessel. The radial artery is
because it is most likely contaminated with IV fluid the primary arterial site, while there may be times
and only subsequent sample tubes should be used when the brachial or femoral artery may be consid-
for analytic purposes. ered. Arterial punctures are more difficult to p ­ erform
 Specimen Collection and Handling 31

Before separation... Separation

SUPERNATANT
Anticoagulant present—plasma
Whole blood contains fibrinogen
(if anticoagulant
present)

Buffy coat

Red Blood Cells

A B
Figure 1.16 Blood sample. (A) Whole blood. (B) Whole blood after separation.
© Wolters Kluwer.

because of inherent arterial pressure, difficulty in causes a stasis of blood flow and an increase in hemo-
stopping bleeding afterward, and the undesirable concentration and anything bound to proteins or
development of a hematoma, which cuts off the blood the cells. Having patients open and close their hand
supply to the surrounding tissue.23 during phlebotomy is of little value and may cause
Continued metabolism may occur if the serum an increase in potassium or lactic acid and, therefore,
or plasma remains in contact with the cells for any should be avoided. IV contamination should be con-
period. Evacuated tubes may incorporate gel-like sidered if a large increase occurs in the substances
material that serves as a barrier between the cells and being infused, such as glucose, potassium, sodium,
the plasma or serum and seals these compartments and chloride, with a decrease of other analytes such
from one another during centrifugation. Some gels as urea and creatinine. In addition, the proper anti-
can interfere with certain analytes, and manufacturer septic must be used. Isopropyl alcohol wipes, for
recommendations should be followed. example, are used for cleaning and disinfecting the
Proper patient identification is the first step collection site; however, isopropyl alcohol is not rec-
in sample collection. The importance of using the ommended for disinfecting the site when drawing
proper collection tube, avoiding prolonged tourni- blood alcohol levels (in such cases, chlorhexidine is
quet application, drawing tubes in the proper order, used as the disinfectant).
and proper labeling of tubes cannot be stressed Blood is not the only sample analyzed in the
strongly enough. Prolonged tourniquet application clinical chemistry laboratory. Urine is the next most

CASE STUDY 1.2, PART 3

Recall Mía, the new graduate.
4. Which of the chemistry specimens provides the fastest turn-around time?
5. The whole blood analysis was performed and resulted. When Mía opened the centrifuge,
she noticed that the specimens were grossly hemolyzed. What should Mía do?

© Ariel Skelley/DigitalVision/Getty Images.

32 Chapter 1 Basic Principles and Practices of Clinical Chemistry

common fluid for determination. Most quantitative tube(s) with the appropriate test requisition and
analyses of urine require a timed sample (usually patient ­identification labels. This is a particularly
24 hours); a complete sample (all urine collected sensitive area of preanalytic error. Bar code labels
within the specified time) can be difficult because (either as 1D linear barcodes, or 2D QR barcodes) or
many timed samples are collected by the patient in radiofrequency ID chip labeling on primary sample
an outpatient situation. Creatinine analysis is often tubes are vital in detecting errors and to minimizing
used to assess the completeness of a 24-hour urine clerical errors. The laboratorian must also ascertain
sample because creatinine output is relatively free if the sample is acceptable for further processing.
from interference and is stable, with little change in The criteria used depends on the test involved but
output within individuals. The average adult excretes usually include volume considerations (i.e., is there
1 to 2 g of creatinine per 24 hours. Urine volume dif- sufficient volume for testing needs?), use of proper
fers widely among individuals; however, a 4-L con- anticoagulants or preservatives (i.e., was it collected
tainer is adequate (average output is ≈2 L). It should in the correct evacuated tube?), whether timing is
be noted that this analysis differs from the creatinine clearly indicated and appropriate for timed testing,
clearance test used to assess glomerular filtration rate, and whether the specimen is intact and has been
which compares urine creatinine output with that in properly transported (e.g., on ice, within a reason-
the serum or plasma in a specified time interval and able period, protected from light). Unless a whole
urine volume (often correcting for the surface area). blood analysis is being performed, the sample is then
Other body fluids analyzed by the clinical chem- centrifuged as previously described and the serum
istry laboratory include cerebrospinal fluid (CSF), or plasma should be separated from the cells if not
paracentesis fluids (pleural, pericardial, and perito- analyzed immediately. Today, the use of serum sep-
neal), and amniotic fluids. The color and characteris- arator tubes and plasma separator tubes is common
tics of the fluid before centrifugation should be noted practice.
for these samples. Before centrifugation, a laborato- Once the sample is processed, the laboratorian
rian should also verify that the sample is designated
should note the presence of any serum or plasma char-
for clinical chemistry analysis only because a single
acteristics such as hemolysis and icterus (increased
fluid sample may be shared among several depart-
bilirubin pigment) or the presence of turbidity often
ments (i.e., hematology or microbiology), and cen-
associated with lipemia (increased lipids). (See
trifugation could invalidate other laboratory testing
Table 1.5.) Many analytes are stable at room tem-
in those areas.
perature between 24 to 72 hours. However, if
CSF is an ultrafiltrate of the plasma and is approx-
testing is not to be performed within 8 hours,
imately two-thirds of the plasma glucose value. For
it is recommended that serum and/or plasma be
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 Table 1.5 Examples of Hemolyzed, Icteric,
and Lipemic/Turbid Samples
obtained on the CSF sample. This is also true for
lactate dehydrogenase and protein assays requested Hemolyzed (red)
on paracentesis fluids. All fluid samples should be
handled immediately, without delay between sample
procurement, transport, and analysis.
Amniotic fluid may be used to assess fetal lung
maturity, congenital diseases, hemolytic diseases,
genetic defects, and gestational age. The laboratorian
should verify the specific handling of this fluid with
the manufacturer of the testing procedure(s).

Sample Processing Hemoglobin

When samples arrive in the laboratory, they are mg/dL 0 50 150 250 525
first processed. In the clinical chemistry laboratory,
g/L 0 0.50 1.50 2.50 5.25
this means correctly matching the blood collection
 Specimen Collection and Handling 33

collection variables such as inappropriate needle

Icteric (yellow)
gauge, venipuncture site selection (small veins), and
venous trauma or difficulty in specimen collection.
Along with the release of hemoglobin, other intra-
cellular components may be released, such as potas-
sium, 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.
Lipemia results when the lipid levels of the
Total Bilirubin patient are elevated and, in turn, is visualized as a
creamy or milky appearance to the serum or plasma
mg/dL 0 1.7 6.6 16 30 upon centrifugation. Lipemia can cause a volume
µmol/L 0 29.1 112.9 273.6 513 displacement for some analytes, such as electrolytes,
as well as interference in light-scattering method-
Lipemic (turbid)
ologies 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 interfer-
ence has occurred, many analyzers are capable of
detecting, then estimating, the interferent and the
effect on sample values. This assessment of hemoly-
sis, icterus, and lipemia is known as the HIL index.
Laboratories have the ability to determine values
Intralipid®
to establish the alert values of the HIL indices as a
means of assessing specimen integrity and accept-
mg/dL 0 125 250 500 1000 ability. Table 1.5 illustrates examples of HIL indices
mmol/L* 0 1.41 2.83 5.65 11.83 in serum or plasma.25
*Concentrations based on dilutions of 20% Intralipid® (or the equivalent).
CLSI C56A_2012 Hemolysis, Icterus, and Lipemia_Turbidity Indices as Indicators of Interference Sample Variables
in Clinical Laboratory Analysis, 1st Edition.
Sample variables (Table 1.6) include physiologic
All photos used with permission from Siemens Medical Solutions USA, Inc.
considerations, proper patient preparation, and
problems in collection, transportation, processing,
and storage. Although laboratorians must include
r­ efrigerated between 2°C and 8°C.24 It is important mechanisms to minimize the effect of these variables
to avoid exposing samples that are light sensitive, on testing and must document each preanalytic inci-
such as ­bilirubin, to artificial or ultraviolet light dent, it is often difficult to control the variables that
for extended periods of time.24 Separated serum involve individuals outside of the l­aboratory. The
and/or plasma may be frozen at −20°C and stored best course of action is to critically assess or antic-
for longer periods without deleterious effects on the ipate variances, identify potential problems, and
results. Repeated cycles of freezing and thawing, implement an action plan that contains policies,
like those that occur in so-called frost-free freezers, procedures, and checkpoints throughout the test-
should be avoided. ing process. Good communication with all person-
Hemolysis can be visually observed in a centri- nel involved helps ensure that the right specimen at
fuged patient sample as a red color due to the release the right time for the right patient is collected each
of hemoglobin. There are patient conditions where and every time to meet the needs of the healthcare
this may occur in vitro, such as hemolytic anemia, team including the patient, laboratory, and ordering
but hemolysis can also be present due to preanalytic physician. Most accreditation agencies require that
34 Chapter 1 Basic Principles and Practices of Clinical Chemistry

Table 1.6 Variables Affecting Select Chemistry Analytes

Physiological factors Diet Samples requiring fasting (glucose)
Medication or herbal Possible interference with analytical methods (biotin)
supplements
Circadian rhythm Analyte changes based on diurnal variation (cortisol)
Timing of collection Serial testing of an analyte (cardiac
troponin) or timing based on medication
(therapeutic drug monitoring)
Patient posture Shift of hemodynamic fluid volume (proteins)
Patient preparation Fasting Proper instructions for 8- to 12-hour fasting
factors
24-hour urine collection Proper instructions for collection
Collection and sample Venipuncture technique Needle selection, site selection to decrease opportunity of
processing factors hemolysis
Tube selection Appropriate sample tube, inversion following collection,
appropriate clotting time
Tourniquet use Prolonged use affects analytes (K+, lactic acid)
Specimen transport and Protection from light (bilirubin) or collect and store on ice
storage (arterial blood gas, ammonia)

© Jones & Bartlett Learning.

l­aboratories consider all aspects of preanalytic varia- separation of cells from serum, improper storage,
tion as part of their quality assurance plans, includ- and collection.
ing effective problem solving and documentation.
Physiologic variation refers to changes that occur
within the body, such as cyclic changes (diurnal or
Chain of Custody
circadian variation) or those resulting from exer- When laboratory tests are likely linked to a crime
cise, diet, stress, gender, age, underlying medical or accident, they become forensic in nature. In
conditions (e.g., fever, asthma, and obesity), drugs, these cases, documented specimen identification is
or posture. Samples may be drawn on patients required at each phase of the process. Each facility has
who are fasting (usually overnight for at least its own forms and protocols; however, the patient,
8 hours). When fasting, many patients may drink and usually a witness, must identify the sample. The
water to avoid becoming dehydrated, which can sample should be collected and then sealed with a
lead to falsely elevated electrolyte results. Patient tamper-proof seal. Any individual in contact with
­preparation for timed samples or those requiring the sample must document receipt of the sample, the
specific diets or other instructions must be well condition of the sample at the time of receipt, and
written and verbally explained to patients. Elderly the date and time it was received. In some instances,
patients often misunderstand or are overwhelmed one witness verifies the entire process and cosigns
by the directions given to them. Collection and pro- as the sample moves along. Any analytic test could
cessing variations are related to those factors dis- be used as part of legal testimony; therefore, the
cussed under specimen processing. Clerical errors ­laboratorian should give each ­sample—even without
are the most frequently encountered, followed by the documentation—the same attention given to a
other pre-­analytical variables including inadequate forensic sample.
 Specimen Collection and Handling 35

Electronic and Paper Reporting a given test based on the reason and type of testing,
of Results and there are codes given for common profiles or
array of tests that represent each test’s separate codes.
Electronic transmission of laboratory data and the For example, blood glucose testing includes the
use of physician order entry, electronic medical codes 82947 (quantitative except for strip reading),
record, coding, billing, and other data management 82948 (strip reading), and 82962 (self-monitoring
systems maintain the integrity of data generated by FDA-cleared device), and the comprehensive
by providing reporting guidelines and safeguards metabolic panel (80053) includes albumin, alkaline
to ensure privacy of the data and records. There phosphatase, total bilirubin, blood urea nitrogen,
are various data management systems in use by total calcium, carbon dioxide, chloride, creatinine,
healthcare agencies related to accessing ­laboratory glucose, potassium, total protein, sodium, and ala-
information. For example, the Logical Observa- nine and aspartate transaminases and their associ-
tion Identifiers Names and Codes (LOINC) system, ated codes. At a minimum, any laboratory reporting
International Federation of Clinical Chemistry/­ system must include a unique patient identifier, test
International Union of Pure and Applied Chemistry name, and code that relates back to the HCPCS and
(IFCC/IUPAC), ASTM, Health Level Seven Interna- ICD databases. For reporting purposes, whether
tional (HL7), and Systematized Nomenclature of paper or electronic, the report should include the
Medicine, Clinical Terms (SNOMED CT) are data- unique patient identifier and test name (including
bases that use unique coding systems for laboratory any appropriate abbreviations), the test value with
observations. There are also additional proprietary the unit of measure, date and time of collection,
systems in use. To standardize these processes and sample information, reference ranges, plus any
to protect the confidentiality of patient information other pertinent information for proper test inter-
as required by the Health Insurance Portability and pretation. Results that are subject to autoverifica-
Accountability Act (HIPAA), the Healthcare Com- tion should be indicated in the report. Table 1.7 lists
mon Procedure Coding System Level II (HCPCS) the information that is often required by accredita-
test and ­services coding system was developed by tion agencies.26
the ­Centers for Medicare and Medicaid Services
(CMS) to be recognized by all insurers for reim-
bursement purposes. The International Classifi-
cation of Diseases (ICD) developed by the World Table 1.7 Minimum Elements of Paper
or Electronic Patient Reports
Health Organization (WHO) uses codes identify-
ing patient diseases and conditions. In the United Name and address of laboratory performing the
States, ICD-11 is currently in place. The clini- analysis including any reference laboratories used
cal modifications are maintained by the National Patient name and identification number or unique
Center for Health Statistics. Incorporated into the identifier
HCPCS system is the Current Procedural Termi-
Name of physician or person ordering the test
nology (CPT) codes, developed by the American
Medical Association, which identify almost all lab- Date and time of specimen collection
oratory tests and procedures. The CPT codes are Date and time of release of results (or available if
divided into different subcategories, with tests or needed)
services assigned five-digit numbers followed by
Specimen source or type
the name of the test or service. Together, these stan-
dard coding systems help patient data and tracking Test results and units of measure if applicable
of disease transmission between all stakeholders
Reference ranges, when available
such as physicians, patients, epidemiologists, and
insurers. Comments relating to any sample or testing
Clinical laboratory procedures are found in CPT interferences that may alter interpretation
Category I with coding numbers falling between © Jones & Bartlett Learning.
80,000 and 89,999. There can be several codes for
36 Wrap-Up Basic Principles and Practices of Clinical Chemistry

WRAP-UP
To support your learning, review
the chapter learning objectives and
complete the online activities. The
Navigate 2 Advantage Access included
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offers a wealth of resources. These
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References
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