CHAPTER 24 Electrolytes and Blood Gas es 413
Ke y Wo rd s a n d De fin it io n s c o n t d
Cystic f brosis (CF) Inherited disorder o a transmembrane con-
ductance regulator protein (CFTR) that leads to chronic pancre-
atic and obstructive pulmonary disease.
Cystic f brosis transmembrane conductance regulator
(CFTR) Atransmembrane protein produced by the CFTR gene.
Electrolytes Charged low-molecular-mass molecules present in
plasma and cytosol, usually ions o (1) sodium, (2) potassium,
(3) calcium, (4) magnesium, (5) chloride, (6) bicarbonate, (7)
phosphate, (8) sul ate, and (9) lactate.
Electrolyte exclusion e ect Electrolytes are excluded rom the
raction o total plasma volume that is occupied by solids,
which leads to underestimation o electrolyte concentration by
some methods.
Hemoglobin (Hb) An oxygen-carrying, heme-containing protein
abundant in red blood cells.
Henderson-Hasselbalch equation Equation that de nes the rela-
tionship between pH, bicarbonate, and the partial pressure o
dissolved carbon dioxide gas:
pH = pK + log cHCO 3
( + PCO3 )
Ion-selective electrode (ISE) Atype o special-purpose, potentio-
metric electrode consisting o a membrane selectively perme-
able to a single ionic species. The potential produced at the
membranesample solution inter ace is proportional to the
logarithm o the ionic activity or concentration.
Osmolal gap A di erence between the observed and calculated
osmolalities in serum analysis. The calculated osmolar values
include sodium concentration multiplied by 1.86, plus glucose
and blood urea nitrogen, plus 9.
Maintenance o water homeostasis is paramount to li e or all
organisms. In humans, the maintenance o water homeostasis
is primarily a unction o the our major electrolytes: Na+, K+,
Cl, and HCO 3. T ese electrolytes also have a role in acid-
base balance and muscle unction, as well as serving as co ac-
tors or enzymes. Abnormal electrolyte concentrations may be
the cause or the consequence o a variety o medical disorders.
Because o their physiological and clinical interrelationships,
this chapter discusses determination o (1) electrolytes, (2)
osmolality, (3) sweat testing, (4) blood gases and pH, and (5)
blood oxygenation.
Ele c tro lyte s
Electrolytes may be classi ed as anions, negatively charged
ions that move toward an anode, or cations, positively charged
ions that move toward a cathode. Important physiological elec-
trolytes include Na+, K+, Ca2+, Mg2+, Cl, HCO 3, H 2PO 4, and
HPO 2 + +
4 . T e major electrolytes (Na , K , Cl , HCO 3 ) occur
primarily as ree ions, whereas signi cant amounts (>40%) o
Ca2+, Mg2+, and trace elements are bound by proteins, mainly
Osmometry Technique or measuring the concentration o dis-
solved solute particles in a solution.
Osmotic pressure The pressure required to stop osmosis through
a semipermeable membrane between a solution and pure
solvent.
Oximetry A technique used to determine the oxygen saturation o
arterial blood.
Oxygen dissociation curve The sigmoidal curve obtained when
S O2 o blood is plotted against P O2.
Oxygen saturation (SO2) The raction (percentage) o unctional
hemoglobin that is saturated with oxygen.
Oxyhemoglobin An hemoglobin that contains bound O2.
P50 P O2 or a given blood sample at which the hemoglobin o the
blood is hal saturated with O2; P50 ref ects the a nity o hemo-
globin or O2.
Partial pressure The substance (mole) raction o gas times the
total pressure; i.e., the partial pressure o oxygen, P O2, is the
raction o oxygen gas times the barometric pressure.
pH The negative logarithm o hydrogen ion activity.
Pilocarpine iontophoresis Noninvasive method that uses elec-
tricity to orce the drug pilocarpine into the skin or the purpose
o inducing sweating at the site.
Point-o -care testing (POCT) Clinical testing that occurs next to
the patient, usually with a handheld device and an unprocessed
specimen collected immediately be ore testing.
Sweat chloride The concentration o chloride in sweat; increased
sweat chloride is characteristic o cystic brosis.
Water homeostasis The body process that maintains a balance o
water intake and output.
albumin. Determination o body uid concentrations o the
our major electrolytes (Na+, K+, Cl, and HCO 3) is com-
monly re erred to as an electrolyte pro le.
Spe c ime ns fo r Ele c tro lyte De te rminatio n
Serum and plasma are the specimens typically analyzed or
their electrolyte content. Capillary blood is another sample
commonly analyzed. Heparinized whole blood arterial or
venous specimens obtained or blood gas and pH determina-
tions may also be used with direct ion-selective electrodes
(ISEs). Di erences in values between serum and plasma and
between arterial and venous samples have been documented,
but only the di erence between serum and plasma K+ is
considered clinically signi cant. Heparin, either the lithium
or ammonium salt, is required i plasma or whole blood is
assayed. Use o plasma or whole blood provides the advan-
tage o shortening turnaround time because it is not necessary
to wait or the blood to clot. Furthermore, plasma or whole
blood provides a distinct advantage in determining K+ con-
centrations, which are invariably higher in serum depending
on platelet count.13 Grossly lipemic blood can be a source o
414 PART III Analytes
analytical error (see Electrolyte Exclusion E ect, later in this
chapter), with some methods requiring ultracentri ugation
o lipemic serum or plasma be ore analysis. Hemolysis o red
blood cells will cause erroneously high K+ results. In addition,
unhemolyzed specimens that are not promptly processed may
have increased K+ concentrations because o K+ leakage rom
red blood cells when whole blood is stored at 4 C.
Collection o urine specimens or Na+, K+, or Cl assays
should be done without the addition o preservatives. Other
types o specimens used or electrolyte assays include (1) body
uid aspirates, (2) eces, (3) gastrointestinal uid, and (4)
sweat samples.
So dium
Sodium is the major cation o extracellular uid. Because it
represents approximately 90% o the 154 mmol o inorganic
cations per liter o plasma, Na+ is responsible or almost one-
hal the osmotic strength o plasma. T e daily diet o the adult
male in the United States contains 3 to 6 g (90 to 250 mmol) o
Na+ (7 to 14 g o NaCl), which is nearly completely absorbed
rom the GI tract.3 T e body requires only 1 to 2 mmol/d, and
the excess is excreted by the kidneys, which are the ultimate
regulators o the amount o Na+ (and thus water) in the body.
T e processes by which the kidneys regulate sodium balance
are discussed in detail in Chapter 35.
Sp e c ime ns
For sodium analysis, (1) serum, (2) plasma, and (3) urine
specimens may be stored at 4 C or may be rozen. Erythro-
cytes contain only one-tenth o the Na+ present in plasma, so
hemolysis does not cause signi cant errors in serum or plasma
Na+ values. Lipemic samples should be ultracentri uged and
the in ranatant analyzed unless a direct ISE is used (see Elec-
trolyte Exclusion E ect).
De te rmina tion o Sod ium in Bod y Fluid s
Sodium may be determined (1) by atomic absorption spectro-
photometry (AAS), (2) electrochemically with an Na+-ISE, or
(3) spectrophotometrically. oday, ISE methods are by ar the
most common. Because sodium and potassium are routinely
assayed together, methods or their analysis are described
together later in this chapter.
Re e re nc e Inte rva ls
A typical re erence interval or serum Na+ is 136 to 145 mmol/L.
T e interval or premature newborns at 48 hours is 128 to 148
mmol/L, and the value or umbilical cord blood rom ull-term
newborns is 127 mmol/L. Urinary sodium excretion varies
with dietary intake, but or an adult male on an average diet
containing 7 to 14 g o NaCl per day, an interval o 120 to 240
mmol/d is typical. A large diurnal variation in Na+ excretion
has been noted, with the rate o Na+ excretion during the night
being only 20% o the peak rate during the day.
Po tas s ium
Potassium is the major intracellular cation. In tissue cells, its
average concentration is 150 mmol/L, and in erythrocytes, the
concentration is 105 mmol/L. High intracellular concentra-
tions are maintained by the Na+, K+ adenosine triphosphate
(A P)ase pump, which is ueled by oxidative energy and con-
tinually transports K+ into the cell against a concentration
gradient. Di usion o K+ out o the cell into the extracellu-
lar uid (ECF) and plasma occurs whenever pump activity
is decreased.10 T e importance o these considerations or
sample integrity in analysis o K+ is discussed later. T e body
requirement or K+ is satis ed by an average dietary intake o
2.4 to 4.4 g/d (60 to 120 mmol/d). Potassium absorbed rom
the gastrointestinal tract is rapidly distributed, and a small
amount is taken up by cells, with most excreted by the kidneys.
Sp e c ime ns
Comments made earlier on specimens or Na+ analysis are
generally applicable to those or K+ analysis. However, potas-
sium concentrations in plasma and whole blood are 0.1 to 0.7
mmol/L lower than those in serum, and most re erence inter-
vals or serum K+ are 0.2 to 0.5 mmol/L higher than those or
plasma K+. T e extent o this di erence depends on the plate-
let count, because additional K+ in serum is primarily a result
o platelet rupture during coagulation.13 T is variability in the
amount o additional K+ in serum makes plasma the specimen
o choice and emphasizes the necessity o noting on reports
whether serum or plasma was assayed and using the appropri-
ate re erence interval.
Specimens or determining K+ concentrations in serum or
plasma must be collected by methods that minimize hemolysis
because release o K+ rom as ew as 0.5% o erythrocytes will
increase K+ values by 0.5 mmol/L. An increase in K+ o 0.6%
has been estimated or every 10 mg/dL o plasma hemoglo-
bin (Hb) caused by hemolysis.3 T ere ore it is imperative that
any visible hemolysis be noted with reported K+ values with a
comment that results are alsely elevated. Whenever hemolysis
is suspected, a portion o the specimen should be centri uged
and visually inspected.
Preanalytical errors that are clinically signi cant have been
observed or K+ determinations when blood samples are not
processed in a timely manner expediently. I a whole blood
specimen is maintained at 4 C versus 25 C be ore separation,
glycolysis is inhibited, and the energy-dependent Na+, K+-
A Pase will not maintain the Na+/K+ gradient. An increase in
plasma K+ will occur as a result o K+ leakage rom erythro-
cytes and other cells.
T e opposite e ect, namely, a alsely decreased K+ value,
has been observed when an unseparated sample is stored at 37
C because glycolysis occurs and K+ shi s intracellularly. Even
at room temperature, leukocytosis will initially cause alsely
decreased K+ concentrations. For reliable K+ determinations,
it is recommended to (1) collect blood with heparin, (2) main-
tain it near 25 C, and (3) separate the plasma within minutes
by high-speed centri ugation without cooling. In practical
terms, separation within 1 hour when samples are maintained
at room temperature is unlikely to introduce great error.
Finally, skeletal muscle activity causes K+ ions to move rom
muscle cells into plasma that results in a marked elevation in
plasma K+ values, such as when an upper arm tourniquet is

not released be ore the beginning o blood draw a er a patient
clenches his st repeatedly.8
Re e re nc e Inte rva ls
Reported re erence intervals or serum vary rom 3.5 to 5.1
mmol/L or adults and rom 3.7 to 5.9 or newborns. For
plasma, a requently cited interval is 3.4 to 4.8 mmol/L or
adults. Cerebrospinal uid concentrations are 70% those o
plasma. Urinary excretion o K+ varies with dietary intake,
but a typical interval observed in an average diet is 40 to
90 mmol/d. Fecal excretion has been reported as 18.2 2.5
mmol/d, but in severe diarrhea, gastrointestinal loss may be as
much as 60 mmol/d.
Me thod s or the De te rmina tion o Sod ium
a nd P ota s s ium
Ion-selective electrodes and spectrophotometric assays are the
techniques o choice to measure sodium and potasium.
Ion-S elective Electrodes
Analyzers connected to ISEs usually contain Na+ electrodes with
glass membranes and K+ electrodes with liquid ion exchange
membranes that incorporate valinomycin. T ese are potentio-
metric devices (see Chapter 10) that determine the change in
electromotive orce (E, potential) in a circuit between a mea-
surement electrode (the ISE) and a re erence electrode, as the
selected ion interacts with the membrane o the ISE. Operation-
ally, the measuring system is calibrated by the introduction o
calibrator solutions containing de ned amounts o Na+ and
K+. T e potentials o the calibrators are determined, and the
E/ log concentration responses are stored in microprocessor
memory as a comparison or calculating unknown concentra-
tion when E o the unknown is measured. Frequent calibration,
initiated by the user or by microprocessor-controlled uptake
o sample rom a reservoir o the calibrator, is typical o most
current ISE systems. Some instruments, particularly point-o -
care testing (POCT) devices and many blood gas analyzers, are
designed to measure Na+ and K+ in whole blood.
Both indirect and direct methods are used with ISE elec-
trodes. With indirect ISE methods, the sample is introduced
into the measurement chamber a er mixing with a large vol-
ume o diluent. Indirect ISE methods are used most commonly
on todays automated, high-throughput clinical chemistry sys-
tems. With direct ISE methods, the sample is presented to the
electrodes without dilution. T is approach became possible
with the miniaturization o electrodes. Direct ISEs are most
common in blood gas analyzers and point-o -care devices in
which whole blood is directly presented to the electrodes.
Errors observed in the use o ISEs are due to (1) lack o
selectivity, (2) repeated protein coating o the ion-sensitive
membranes, or (3) contamination o the membrane or salt
bridge by ions that compete or react with the selected ion and
thus alter the electrode response.
S pectrophotom etric Methods
Spectrophotometric methods all into two categories: (1)
those based on enzyme activation, and (2) those that detect
CHAPTER 24 Electrolytes and Blood Gas es 415
the spectral shi produced when Na+ or K+ binds to a macro-
cyclic chromophore. T e high cost o reagents or these meth-
ods and the act that ew problems are associated with ISE
methods have resulted in small niche use o these methods,
primarily with smaller instruments used in physicians o ces
or clinics.
Ele c trolyte Exc lus ion E e c t 1
T e electrolyte exclusion e ect describes the exclusion o elec-
trolytes rom the raction o the total plasma volume that is
occupied by solids. T e volume o total solids (primarily pro-
tein and lipid) in an aliquot o plasma is approximately 7%, so
that 93% o plasma volume is actually water. T e main elec-
trolytes (Na+, K+, Cl, and HCO 3) are con ned to the water
phase. When a xed volume o total plasma (e.g., 10 L) is
pipetted or dilution be ore indirect ISE analysis, only 9.3 L
o plasma water that contains the electrolytes is added to the
diluent. T us a concentration o Na+ determined by indirect
ISE to be 140 mmol/L is the concentration in the total plasma
volume, not in the plasma water volume. In act, i the plasma
contains 93% water, the concentration o Na+ in plasma water
is [140 (100/93)], or 150 mmol/L. T is negative error in
plasma electrolyte analysis has been recognized or many
years. Even though it is the electrolyte concentration in plasma
water that is physiological (the Na+ concentration o normal
saline is indeed 150 mmol/L), it was assumed that the volume
raction o water in plasma is su ciently constant that this
di erence could be ignored. In act, all electrolyte re erence
intervals are based on this assumption and actually re ect con-
centrations in total plasma volume, not in water volume. Indeed,
virtually all concentrations measured in the clinical chemistry
laboratory are related to the total sample volume rather than
to the water volume. T is electrolyte exclusion e ect becomes
problematic when pathophysiological conditions are present
that alter the plasma water volume, such as hyperlipidemia or
hyperproteinemia. In these settings, alsely low electrolyte val-
ues are obtained whenever samples are diluted be ore analysis,
as with an indirect ISE method (Figure 24-1).1
It is the dilution o total plasma volume and the assumption
that plasma water volume is constant that render the indirect
ISE the method subject to the electrolyte exclusion e ect. Direct
ISE methods still determine the concentration relative to activ-
ity but do not require sample dilution. Because no dilution
occurs, activity is directly proportional to the concentration in
the water phase, not the concentration in the total volume. o
ensure that results rom direct ISEs are equivalent to those rom
indirect ISEs, most direct ISE methods actually operate in what
is commonly re erred to as the ame mode.* In this mode, the
directly measured concentration in plasma water is multiplied
by the average water volume raction o plasma (0.93). Although
the latter may vary widely, as long as the activity o the speci c
ion is constant, the concentration o the ion in the water phase
*In the ame mode, results were obtained with the traditional technique o
ame photometry. Results or these analytes were expressed in terms o sub-
stance concentration. In current practice, the technique o ame photometry
is no longer used in clinical laboratories.
416 PART III Analytes
100
80
e
m
60 100% 90% 80%
u
H2 O H2 O H2 O
l
o
V
40
20
0
Dire ct pote ntiome try 100 100 100
Fla me photome try 100 90 80
or
Indire ct pote ntiome try
Fig ure 24-1 Predicted in uence o water content on sodium
measurements or a 100 mmol/L NaCl solution by direct ion-
selective electrode (ISE) versus ame emission photometry or
indirect ISE. Red areas represent nonaqueous volumes, which
could consist o lipids, proteins, or even a slurry o latex or sand
particles. (Adapted rom Apple FS, Koch DD, Graves S,
Ladenson J H. Relationship between direct-potentiometric and
ame-photometric measurement o sodium in blood. Clin
Chem 1982;28:1931-5.)
becomes independent o the relative proportions o water and
total solids i the ion is not bound by proteins. T ere ore direct
ISE methods are ree o electrolyte exclusion e ects.
Most clinical chemists and physicians have reached the
conclusion that direct ISE methods or electrolyte analysis are
the methods o choice. However, it is clear that results rom
direct methods will continue to be converted to total plasma
volume concentrations by use o the ame mode, and indeed
this is the recommendation o the Clinical Laboratory and
Standard Institute (CLSI). able 24-1 and able 24-2 summa-
rize methods that are and are not subject to electrolyte exclu-
sion e ects, respectively.
Chlo ride
Chloride is the major extracellular anion; like Na+, it is signi -
cantly involved in the maintenance o (1) water distribution,
(2) osmotic pressure, and (3) anion-cation balance in the ECF.
In contrast to its high ECF concentrations (103 mmol/L), the
concentration o Cl in the intracellular uid o erythrocytes is
45 to 54 mmol/L, and only 1 mmol/L in the intracellular uid
in most other tissue cells. Chloride ions are almost completely
absorbed rom the intestinal tract and excreted by the kidneys.
Sp e c ime ns
Chloride most o en is measured in (1) serum. (2) plasma, (3)
urine, or (4) sweat. Even gross hemolysis does not signi cantly
alter serum or plasma Cl concentration because the erythrocyte
concentration o Cl is approximately hal that o plasma. Because
very little Cl is protein bound, change in posture or stasis, or the
use o tourniquets, has little e ect on its plasma concentration.
Me thod s or De te rmina tion o Chlorid e in Bod y Fluid s
Historically, chloride was measured in body uids and solids
by mercurimetric titration and spectrophotometric methods.
TABLE 24-1 Me thod s Me as uring Conc entra tion in
the Whole Sa mple Volume and Thus
Subjec t to Ele ctrolyte Exclus ion E ect
Method Analytes
Atomic absorption spectrometry Ca2+ , Mg2+ , and others
Amperometry/coulometry Cl
Indirect potentiometry Na+, K+, Ca2+ , Cl
TABLE 24-2 Me thod s Me a s uring Ac tivity, Mola lity,
or Conc e ntra tion in the Wa te r P ha s e
a nd Thus Not Sub je c t to Ele c trolyte
Exc lus ion E e c t
Method Analytes
ISEs with undiluted sample H+ (pH), Na+ , K+, Ca2+, Cl, Li+
Gas electrodes CO2 (P CO2), O2 (P O2)
HCO3 (calculated rom pH and P CO2)
Freezing point depression H2O (osmolality)
ISE, Ion-selective electrode.
As these methods are no longer used, coulometric-ampero-
metric titration and ISEs are now the methods o choice
Coulom e tric-Am perom etric Titration
Reactions in coulometric-amperometric determinations o
Cl depend on the generation o Ag+ rom a silver electrode
at a constant rate and on the reaction o Ag+ with Cl in the
sample to orm insoluble silver chloride (AgCl):
Ag + + Cl AgCl
A ter the stoichiometric point is reached, excess Ag+ in
the mixture triggers shutdown o the Ag+ generation sys-
tem. A timing device records elapsed time between the
start and stop o Ag+ generation. Because the time inter-
val is proportional to the amount o Cl in the sample, the
concentration o Cl is then calculated. Applications o
the coulometric-amperometric principle (o ten called the
Cotlove chloridometer technique) are the most precise
methods or measuring Cl over the entire range o concen-
trations ound in body luids.
Ion-S elective Electrode Methods
Solvent polymeric membranes that incorporate quaternary
ammonium salt anion exchangers, such as tri-n-octylpro-
pylammonium chloride decanol, are used to construct Cl-
selective electrodes in clinical analyzers. Although they are
by ar the most common methods o measuring Cl in clini-
cal laboratories, these electrodes o ten su er rom mem-
brane instability and lot-to-lot inconsistency in terms o
selectivity or other anions. Anions that tend to be prob-
lematic include other halides and organic anions, such as
SCN , which are particularly problematic because o their
ability to solubilize in the polymeric organic membrane o
these electrodes.
 CHAPTER 24 Electrolytes and Blood Gas es 417

Ag e
CF ne wbo rn s c re e ning re s ult:
P os itive IRT/DNA or IRT/IRT
5-14 days

Notifica tion of pa re nts a nd P CP

~2 we e ks
CF c e nte r diag no s tic e valuatio n:

S we a t chloride te s t* 2-4 we e ks

60 mmol/L 30-59 mmol/L 29 mmol/L

2 CF 0-1 CF no DNA da ta
muta tions muta tion
Outc o me s :

Dia gnos is of CF P os s ible CF CF ve ry unlike ly

CF ce nte r follow-up: DNA a na lys is 1-2 months

DNA a na lys is if IRT/IRT Us ing CFTR
Clinica l a s s e s s me nts multimuta tion
Be gin the ra py a ime d me thod
to s ta y he a lthy Ancilla ry te s ts
S we a t te s t s iblings

Re pe a t s we a t
2-6 months
chloride te s t

*If the ba by is a t le a s t 2 kg a nd more tha n 36 we e ks ge s ta tion a t birth, pe rform bila te ra l s we a t

s a mpling/a na lys is with e ithe r Gibs on-Cooke or Ma croduct me thod;
re pe a t a s s oon a s pos s ible if s we a t qua ntity is le s s tha n 75 mg or 15 l, re s pe ctive ly.

CF muta tion re fe rs to a CFTR muta nt a lle le known to ca us e CF dis e a s e .

The dis e a s e is ve ry unlike ly; howe ve r, if the re a re 2 CF muta tions in tra ns , CF ma y be dia gnos e d.

Afte r a re pe a t s we a t te s t, furthe r e va lua tion de pe nds on the re s ults a s implie d a bove .
Fig ure 24-2 Diagnostic algorithm or cystic f brosis (CF) a ter newborn screening. (From Far-
rell PM, Rosenstein BJ , White TB, et al; Cystic Fibrosis Foundation. Guidelines or
diagnosis o cystic f brosis in newborns through older adults: Cystic Fibrosis Foundation
consensus report. J Pediatr 2008;153:S4-S14.)

Reference Intervals chronic obstructive pulmonary disease and pancreatic insu -

Reported re erence intervals or Cl in serum or plasma vary-
rom 98 to 107 mmol/L to 100 to 108 mmol/L. Serum val-
ues vary little during the day. Spinal uid Cl concentrations
are 15% higher than those in serum. Urinary excretion o
Cl varies with dietary intake, but an interval o 110 to 250
mmol/d is typical.
Me a s ure me nt o Swe a t Chlorid e (Swe a t Te s ting)
Analysis o sweat or increased chloride concentration is used
to con rm the diagnosis o cystic f brosis (CF). CF is the most
common lethal genetic disorder o the Caucasian population,
with a wide spectrum o clinical presentations, including
ciency. CF is caused by a de ect in the cystic f brosis trans-
membrane conductance regulator protein (CFTR), a protein
that normally regulates electrolyte transport across epithelial
membranes. More than 1500 mutations o CF R have been
identi ed. Although direct mutational analysis is available, it is
not in ormative in all cases, and a quantitative sweat chloride
test remains the standard or diagnostic testing. In an e ort to
standardize testing, the CLSI developed the guidelines docu-
ment C34-A3.5
Sweat testing is o en per ormed in conjunction with new-
born screening programs (Figure 24-2). Newborn screening
or CF occurs throughout the United States and the world.
418 PART III Analytes
Most newborn screening protocols begin with an immunore-
active trypsinogen assay (IR ) rom a dried blood spot and
are ollowed by a second IR or DNA testing.7 In ants with a
positive newborn screening test are re erred or a quantitative
sweat chloride test, which has resulted in an increase in the
number o sweat tests per ormed on individuals younger than
2 months o age.
T e sweat test is per ormed in three phases: (1) sweat
stimulation by pilocarpine iontophoresis, (2) collection o
the sweat, and (3) qualitative or quantitative analysis o sweat
chloride, sodium, or conductivity.
S weat S tim ulation and Colle ction
Because o transient increases in sweat electrolytes shortly a er
birth, individuals should be at least 48 hours old be ore a sweat
chloride test is per ormed. T e child should be (1) physiologi-
cally and nutritionally stable, (2) thoroughly hydrated, and (3)
ree o acute illness. T e skin should be ree o cuts, rashes,
and in ammation to avoid contamination o the sweat sample
with serous uid. For example, sweat testing never should be
per ormed over an area o eczema.
Stimulation. o stimulate sweat, localized sweating is pro-
duced by pilocarpine iontophoresis into an area o the skin.
Iontophoresis uses a small electrical current to deliver pilocar-
pine into the sweat glands rom the positive electrode, while
an electrolyte solution at the negative electrode completes the
circuit. Note: Although the Occupational Sa ety and Health
Administration (OSHA) does not list sweat as potentially
in ectious, laboratory personnel should practice the same uni-
versal precautions they would use with any other body uid.
Collection. A er iontophoresis, sweat is collected onto (1)
preweighed gauze pads, (2) lter paper, (3) Macroduct coils,11
or (4) Nanoduct conductivity sensor cells using techniques to
minimize evaporation and contamination. I sweat is collected
onto gauze or lter paper, the electrodes usually are made
o copper and are slightly smaller than the stimulation and
collection area. T e composition o the electrolyte solution
should be selected to avoid contamination with the sweat sam-
ple. Be ore collection is per ormed, the gauze or lter paper
used or sweat collection should be placed into a weighing vial
with a secure sealing lid, and the vial labeled and weighed with
an analytical balance. For a detailed procedure or stimulation
and collection, the reader should re er to the CLSI document
C34-A3.5
Alternatively or sweat stimulation, the electrodes and
the current source are integrated, as they are in the Wescor
Macroduct and Nanoduct systems (http://www.wescor.com/;
accessed September 30, 2015), which use gel reagents contain-
ing pilocarpine. In the Macroduct system, sweat is collected
in a disposable microbore-tubing coil collector.11 A er su -
cient sweat has been collected, the sweat is trans erred rom
the coil into a sealable microsample cup. T e Nanoduct system
employs an integrated conductivity cell sensor in the single-
use collection device.
Critical Issues Associated With Sweat Stimulation and
Collection. During collection, the analyst must (1) avoid
evaporation and contamination o the sample, (2) collect
a su cient amount o sample, and (3) minimize skin reac-
tions. Determination o and adherence to a minimum sweat
weight or volume are critical to obtain valid sweat testing
results. T e requirement or a minimum amount ensures an
appropriate sweat rate and sweat electrolyte concentration and
is independent o the instrument used to measure sweat elec-
trolytes. Un ortunately, many laboratorians misunderstand
the necessity o collecting the correct volume, leading to alse-
positive and alse-negative sweat tests, which have signi cant
implications or patient care.
Sweat electrolyte concentration is related to sweat rate. At
low sweat rates, sweat electrolyte concentration decreases and
the opportunity or sample evaporation increases. o ensure
a valid result, the average sweat rate should exceed 1 g/m 2/
min. o standardize and simpli y the collection process, the
size o the (1) electrodes, (2) reagent pads, and (3) collection
material must be approximately the same. Insu cient samples
must not be pooled or analysis.
When the acceptable rate is applied to the parameters
described in the CLSI document, the minimum acceptable
sample or analysis rom a single site with use o 2 2-inch
gauze or lter paper or stimulation and collection is 75 mg o
sweat collected within 30 minutes.When the collection pro-
cess deviates rom standard parameters, the minimum accept-
able sweat volume or weight changes, and sweat should be
collected or only 30 minutes. I the collection time exceeds 30
minutes, the requirement or the amount o sweat needed to
ensure adequate stimulation must increase. Extending the col-
lection time allows additional opportunity or sweat evapora-
tion and, practically, does not signi cantly increase the sweat
yield.
Acquiring the minimum sample should not be a problem
with most patients i the the CLSI5 and the manu acturers
recommendations are ollowed. On average, the percentage
o insu cient samples should not exceed 5% or patients
older than 3 months o age and 10% or patients 3 months o
age or younger.5,12 I the laboratory collects sweat rom two
sites (bilateral testing), the test is considered QNS (quantity
not su cient) only when both sites are inadequate. Insu -
cient sweat samples result rom several actors, such as (1)
age, (2) race, (3) skin condition, and (4) hydration status.
Also, collecting an adequate amount o sweat is more chal-
lenging in patients younger than 1 month o age.12 For this
reason, it is recommended that sweat testing in asymptom-
atic individuals be per ormed when the in ant (1) is at least 2
weeks o age, (2) was more than 36 weeks gestation at birth,
and (3) weighs more than 2 kg.5,12 I an adequate sweat sam-
ple is not obtained, the test should be repeated as soon as is
practical.
Burns to the patients skin a er iontophoresis are extremely
rare but have occurred at either electrode. I the burn occurs
at the site o pilocarpine stimulation, sweat should not be
collected.
Qualitative Tests
A qualitative sweat test represents a screening test or CF.
Individuals who have positive or borderline results should

then undergo quantitative sweat chloride testing. Examples
o screening tests include Wescor Sweat-Chek and Nanoduct
or conductivity. Screening tests may or may not measure the
amount o sweat collected and may report a result as posi-
tive, negative, or borderline or give an actual concentration
o sweat analytes. Although a variety o systems are used or
sweat testing, problems have been documented or several
methods, making them inappropriate or clinical use. For
example, older conductivity analyzers using unheated col-
lection cups and direct application chloride electrodes are
not recommended as diagnostic procedures because prob-
lems have been reported with (1) sample evaporation, (2)
condensation, and (3) the ability to quanti y sweat samples
adequately.
he Cystic Fibrosis Foundation has approved the Wes-
cor Macroduct Sweat-Chek or screening at clinical sites,
such as community hospitals, using the criterion that an
individual who has a sweat conductivity o 50 mmol/L or
greater should be re erred to an accredited CF care center
or a quantitative sweat chloride test. Note that sweat con-
ductivity methods produce values that are approximately
15 mmol/L higher than those associated with sweat chlo-
ride concentration. Because o this di erence, laborato-
ries should not report conductivity results as i they were
chloride results. In addition to conductivity results (in
mmol/L), the report should include sweat conductivity re -
erence intervals.
Quantitative Tests
T e diagnosis o CF includes a quantitative measurement o
sweat chloride, which consists o (1) collection o sweat into
gauze, lter paper, or Macroduct coils; (2) evaluation o the
amount collected in weight (milligrams) or volume (microli-
ters); and (3) subsequent measurement o the sweat chloride
concentration.9 In general, chloride concentration is deter-
mined by coulometric titration with a chloridometer. I a labo-
ratory chooses to quanti y sweat chloride with an automated
analyzer that employs an ISE, these methods must be validated
systematically or accuracy, precision, and lower limit o detec-
tion. For any given method, the lower limit o the analytical
measurement range or sweat chloride on unadulterated sweat
should be less than or equal to 10 mmol/L.
Re e re nc e Inte rva ls or Swe a t Chlorid e
Re erence intervals or sweat chloride must be strati ed by
patient age.9
Infants
For in ants 6 months o age or younger, the ollowing re erence
intervals are recommended.9
29 mmol/L: CF unlikely
30 to 59 mmol/L: intermediate
60 mmol/L: indicative o CF
However, as data continue to be generated rom newborn
screening programs, these re erence intervals may have to be
adjusted.
CHAPTER 24 Electrolytes and Blood Gas es 419
Beyond Infancy
For individuals older than 6 months, the ollowing re erence
intervals are recommended9:
39 mmol/L: CF unlikely
40 to 59 mmol/L: intermediate chance o CF
60 mmol/L: indicative o CF
A healthy sweat chloride concentration alone is insu cient
to rule out the diagnosis; it should be interpreted in light o
the clinical picture and with the knowledge that normal con-
centrations have been associated with CF. Several mutations o
the CF gene are associated with intermediate or normal sweat
chloride concentrations.
Qua lity As s ura nc e
Laboratories that provide high-quality sweat testing should
(1) select appropriate methods, (2) have su cient testing
volumes to ensure amiliarity with the test, and (3) limit test-
ing personnel to a small number o well-trained individu-
als. o monitor the accuracy and precision o the analytical
process, two concentrations o controls should be per ormed
daily when patient samples are analyzed. Sweat chloride con-
centrations greater than 160 mmol/L are not physiologically
possible and represent specimen contamination or analytical
error. An important part o a quality assurance plan includes
method validation and external validation o sweat analysis
accuracy through participation in pro ciency testing, such as
that o ered by the College o American Pathology (CAP). T e
reader is re erred to re erence 12 or additional suggestions on
developing a robust quality assurance program or sweat chlo-
ride testing and techniques to minimize QNS collections.
Bic arbo nate (To tal Carbo n Dio xide )
otal carbon dioxide is measured in the clinical laboratory by
(1) acidi cation o a serum or plasma sample and measure-
ment o carbon dioxide released by the process or (2) alkalini-
zation and measurement o total bicarbonate.
Sp e c ime ns
T e same sample types used or Na+ or K+ may be assayed.
Given a specimen in a vacuum draw tube, the concentration
o total CO2 is most accurately determined when the assay is
done as promptly as possible a er collection and centri uga-
tion o the blood in the unopened tube. Ambient air contains
ar less CO2 than does plasma, and dissolved CO2 will escape
rom the specimen into the air, with a consequent decrease in
the CO2 value o up to 4 to 5 mmol/L in the course o 1 hour.
Me thod s or De te rmina tion o Se rum
or P la s ma Tota l Ca rb on Dioxid e
Methods used or total CO 2 measurement with todays auto-
mated instruments may be electrode based or enzymatic. With
indirect electrode-based methods, the amount o released
gaseous CO2 a er acidi cation is determined by a PCO 2 elec-
trode. Direct ISE methods or total CO2 are no longer com-
mon on automated analyzers because o problems with speci-
city. With enzymatic methods or CO2, the specimen is rst
420 PART III Analytes
alkalinized to convert all CO 2 and carbonic acid to HCO 3. T e
enzymatic reactions lead to decreased absorbance o NADH at
340 nm that is proportional to the total CO2 content.
O O
2 2
O P O P hos phoe nolpyruva te
2 ca rboxyla s e
O O HCO 3
H2 C C C
2 C
O O
P hos phoe nolpyruva te
Oxa loa ce ta te
O O
2
O O
C Ma la te
de hydroge na s e
C O HO
CH2
C 2
NADH NAD
O O H O
Oxa loa ce ta te
Re e re nc e Inte rva ls
T e re erence interval or total carbon dioxide in healthy
adults is 22 to 30 mmol/L but is method dependent.
Princ iple s o f Os mo tic Pre s s ure and Os mo s is
Osmometry is a technique or measuring the concentration o
solute particles that contribute to the osmotic pressure o a solu-
tion. Osmotic pressure governs the movement o solvent (water
in biological systems) across membranes that separate two solu-
tions. Di erent membranes vary in pore size and thus in their
ability to select molecules o di erent size and shape. Examples o
biologically important selective membranes are those enclosing
the glomeruli o the kidney and capillary vessels that are perme-
able to water and to essentially all small molecules and ions, but
not to larger protein molecules. Di erences in the concentrations
o osmotically active molecules that are unable to cross a mem-
brane cause those molecules that are able to cross the membrane
to move to establish an osmotic equilibrium. T is movement o
solute and permeable ions exerts osmotic pressure.
Co llig ative Pro pe rtie s
In addition to increasing osmotic pressure when the solute
is added to the solvent, the vapor pressure o the solution
is lowered below that o the pure solvent. As a result o the
change in vapor pressure, the boiling point o the solution
is raised above and the reezing point o the solution is low-
ered below that o the pure solvent. T ese our properties o
solutions(1) increased osmotic pressure, (2) lowered vapor
pressure, (3) increased boiling point, and (4) decreased reez-
ing pointare called colligative properties. All are directly
related to the total number o solute particles per mass o
solvent. T e term osmolality expresses concentrations rela-
tive to mass o the solvent (1 osmolal solution is de ned
to contain 1 Osmol/kg H 2O), whereas the term osmolarity
expresses concentrations per volume o solution (1 osmolar
solution is de ned to contain 1 Osmol/L solution). Osmo-
lality (Osmol/kg H 2O) is a thermodynamically more exact
expression because solution concentrations expressed on a
2
O weight basis are temperature independent, whereas those
C based on volume vary with temperature. Although the term
C O
Pi
osmolarity is o en used in the medical literature, osmolality
CH2 is what the clinical laboratory measures.
An electrolyte in solution dissociates into two (in the case
2
O o NaCl) or three (in the case o CaCl2) particles; there ore
the colligative e ects o such solutions are multiplied by the
number o dissociated ions ormed per molecule. However,
because o incomplete electrolyte dissociation and associa-
2
tions between solute and solvent molecules, many solutions
C
do not behave in the ideal case, and a 1-molal solution may
CH give an osmotic pressure lower than is theoretically expected.
CH2 T e osmotic activity coe cient is a actor used to correct or
C 2 deviation rom the ideal behavior o the system:
O
Ma la te osmol
Osmolality= = nC
Kg H2O
where = osmotic coe cient, n = number o particles into
which each molecule in the solution potentially dissociates,
and C = molality in mol/kg H 2O.
Glucose has an osmotic coe cient o 1.00, whereas the or
sodium chloride is 0.93 at the concentrations ound in serum
thus the derivation o 1.86 Na+ (mmol) in the ormula to
calculate plasma osmolality (NaCl potentially contributes
two osmotically active particles 0.93 = 1.86). Ethanol has an
osmotic coe cient o 0.83. T e total osmolality or osmotic pres-
sure o a solution is equal to the sum o the osmotic pressures or
osmolalities o all solute species present. T e electrolytes Na+,
Cl, and HCO 3, which are present in relatively high concen-
trations, make the greatest contributions to serum osmolality.
Nonelectrolytes such as glucose and urea, which are present nor-
mally at lower molal concentrations, contribute less, and serum
proteins contribute less than 0.5% o the total serum osmolality.
De te rminatio n o f Plas ma and Urine Os mo lality
Determination o plasma and urine osmolality is use ul in the
assessment o electrolyte and acid-base disorders. T e major
osmotic substances in normal plasma are Na+, Cl, glucose,
and urea; thus expected plasma osmolality is calculated rom
the ollowing empirical equation:
mOsmol/kg = 1.86 [Na+ (mmol/L)]
+ glucose [mmol/L]
+ urea [mmol/L]
+9
or
mOsmol/kg = 1.86 [Na+ (mmol/L)]
+ glucose [mg/dL]/18
+ urea N[mg/dL]/2.8
+9