MINI-REVIEW

Interference in Ion-Selective Electrodes Due to

Proteins and Lipids
Sudip Kumar Datta and Parul Chopra*

Background: Ion-selective electrodes (ISE) have become the mainstay of electrolyte measurements in the clini-

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cal laboratory. In most automated analyzers used in large diagnostic laboratories, indirect ISE (iISE) -based electro-
lyte estimation is done; whereas direct ISE (dISE) -based equipment are mostly used in blood gas analyzers and in
the point-of-care (PoC) setting.
Content: Both the techniques, iISE as well as dISE, are scientifically robust; however, the results are often not in-
terchangeable. Discrepancy happens between the two commonly due to interferences that affect the two measur-
ing principles differently. Over the last decade, several studies have reported discrepancies between dISE and iISE
arising due to abnormal protein and lipid contents in the sample.
Summary: The present review endeavors to consolidate the knowledge accumulated in relation to interferences
due to abnormal protein and lipid contents in sample with the principal focus resting on probable solutions
thereof.

IMPACT STATEMENT

• Interference due to abnormal protein and lipid concentrations on electrolyte measurements by Ion-
selective electrodes (ISE) is widely reported.
• This predominantly affects indirect ISE leading to discrepancy between indirect and direct ISE results.
• Several researchers have suggested solutions for correction of indirect ISE results using sample free
water content, algorithms using protein and lipid concentration, or otherwise.
• These solutions have their own limitations and are not universally applicable. Moreover, reports studying
the effects of these interferents simultaneously are few and lack consensus.
• This review aims to consolidate the existing knowledge and probable solutions thereof.

Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
*Address correspondence to this author at: Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
E-mail parul6588@yahoo.co.in.
Received July 13, 2021; accepted September 13, 2021.
DOI: 10.1093/jalm/jfab125
C American Association for Clinical Chemistry 2021. All rights reserved.
V
For permissions, please email: journals.permissions@oup.com.

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MINI-REVIEW
INTRODUCTION
Ion-selective electrodes (ISE) are potentiometric
sensors used to detect the potential difference
between two half cells within an electrochemical
cell. When this potential difference is produced at
a selective membrane and solution interface, it is
proportional to the logarithm of ionic activity or
concentration of the ion being measured. The
membrane may be composed of glass, polymeric
material, or crystalline material; however, it is se-
lective for the ion of interest. Measurements by
ISEs are rapid, have high accuracy and precision,
low cost, use small sample volumes, and provide
measurements over a wide range. Thus, they have
become the mainstay of electrolytes measure-
ment in the clinical laboratory.
ISEs may be direct and indirect. Direct ISEs
(dISE) are present mostly in arterial blood gas
(BGA) and point-of-care (PoC) testing analyzers. In
dISE, the membrane is directly exposed to the
sample, and it measures electrolyte activity.
Measurement by direct potentiometry is depen-
dent only on molality and therefore not affected
by variations in the concentration of protein or lip-
ids in the sample. Indirect ISEs (iISE) are commonly
used in high throughput automated chemistry
analyzers. In iISEs, samples are diluted before test-
ing to allow measurement with lower sample vol-
umes and to expand the measurable
concentration range. In the last 2 decades several
studies have reported the discrepancies arising
between dISE and iISE values due to abnormal
protein and lipid concentrations in the sample.
Dimeski et al. has discussed several interferences
in his grand review published in 2010 (1). Here, we
focus on the discrepancies arising out of abnor-
mal protein and lipid content in the samples and
consolidate the possible corrective measures.
Interference in measurement may occur when
a different substance present in the specimen
leads to a false alteration in measurement leading
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Interference in Ion-Selective Electrodes
to erroneously high or low results. Discrepancy is
the difference in results arising between different
analytic techniques measuring the same analyte.
Interferents in a specimen may lead to discrep-
ancy in measurement due to their variable effect
on the two measuring principles. During estima-
tion of electrolytes by iISE, interference has often
been reported due to high or low concentration
of proteins and lipids, but dISE methods are
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known to be relatively spared (2–4). This often
leads to discrepancies in values obtained from the
two ISE methods. It is important to identify these
interferences and take appropriate steps or es-
tablish procedures to eliminate the effect of such
interferences by either removing the interferent
or using appropriate testing methods.
DISCREPANCIES DUE TO ELECTROLYTE
EXCLUSION EFFECT
A common factor affecting the accuracy of the
iISE method is displacement of plasma water by
proteins and lipids in cases of hyperproteinemia
or hyperlipidemia, by a phenomenon recognized
as the “electrolyte exclusion effect” (5). In iISE,
samples are premixed with diluent, varying in ra-
tios from 1:5 to 1:46 allowing measurement with
lower sample volumes and expanding the mea-
surable concentration range (6). The concentra-
tion of the electrolytes is proportional to molality
and mass concentration of water in kg/L in the
plasma, which is normally taken as 0.93 kg/L for
calculation purposes. However, in samples with el-
evated lipids and/or proteins the value may be as
low as 0.8 kg/L. When the dissolved proteins or lip-
ids increase, iISE output that is standardized for a
predilution plasma water mass concentration of
0.93 kg/L gives falsely low results. This phenome-
non of “pseudohyponatremia” has been reported
in the literature due to the effects of proteins as
well as lipids (7). Techniques requiring sample dilu-
tion, such as in flame photometry or iISE, are
| 07:02 | March 2022
Interference in Ion-Selective Electrodes
reported to be affected by the presence of in-
creased proteins and/or lipids in the sample be-
cause the calculation algorithms assume mass
concentration of water to be 0.93 kg/L (8, 9).
Similarly, studies showing erroneous hypokalemia
and decreased chloride (Cl) concentrations are
also available and are thought to be of similar
magnitude; however, they are less evident in view
of their lower concentrations in serum compared
to sodium (Naþ) (7). On the other hand, in dISE,
the sample comes in direct contact with the selec-
tive membrane and electrolyte estimation is not
affected by any variation in the nonaqueous
phase giving a true estimate of the plasma electro-
lytes despite any changes in nonaqueous plasma
components. Direct ISE provides measurement of
activity, which is the concentration of free un-
bound ions in solution, and it depends on the ac-
tivity coefficient of the sample. Activity coefficient
of the sample, in turn, depends on the ionic
strength. The charge of protein and other macro-
and micromolecules contribute to the ionic
strength of the sample. This fundamental differ-
ence between the two apparently similar technol-
ogies often gives rise to discrepancies in
electrolyte measurement.
The opposite effect of pseudohypernatremia
has also been reported in literature due to low
protein or albumin concentration (10–12). In a re-
cently reported study, almost half of the samples
analyzed using iISE, which were wrongly classified
as normonatremic, were found to be of pseudo
normonatremia, i.e., a normal value obtained
when there is true hyponatremia (13). However,
not enough literature is available on this issue.
Dimeski et al. (14) have defined the clinically rele-
vant difference between indirect and direct so-
dium results based on analytical and biological
variation as 4 mmol/L (14). Using this cutoff, one
study reported that, all five different routine
chemistry instruments using iISE, compared in
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March 2022 |
MINI-REVIEW
that study, produced significant pseudohyperna-
tremia in hypoproteinemic samples (<40 g/L) (13).
Similar findings were reported earlier in another
study, where 34 out of 80 ICU patients (43%)
exceeded a difference of 4 mmol/L between iISE
and dISE (6). Interestingly, different automated
analyzers use different dilutions for electrolytes
measurement. Surprisingly, the equipment using
the least dilution factor was observed to have the
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highest differences from the dISE measurements;
hence the interplay of other factors contributing
to the process needs to be investigated.
Several studies also report the effect of lipemia
on values Naþ, Kþ, and Cl concentrations mea-
sured by iISE vis-à-vis dISE (9, 15). Hyperlipidaemia
decreases the serum electrolyte levels when mea-
sured by iISE in a similar way to hyperproteinemia.
Dimeski et al. (9) observed that there is a de
crease of Naþ and Cl by 1 mmol/L and Kþ by
0.04 mmol/L, respectively, per 10 mmol/L increase
in total lipids i.e., cholesterol plus triglycerides
(TG). Case reports are also available for patients
with obstructive jaundice having hypercholesterol-
emia who presented with pseudohyponatraemia
(16, 17). These articles report patients with chole-
stasis secondary to bone marrow transplant with
graft versus host disease, primary biliary cirrhosis,
hepatitis C, or drug or secondary to structural ab-
normality in the case of pancreatic cancer; all hav-
ing hyperlipidemia and erroneous electrolyte
values (10, 18, 19). In a case report, a 43-year-old
woman with refractory primary biliary cirrhosis
and cholestasis had extreme hypercholesterol-
emia from elevations of lipoprotein-X particles
that resulted in pseudohyponatremia. After multi-
ple plasmapheresis and normalization of choles-
terol level from 2415 mg/dL (62.45 mmol/L) to
200 mg/dL (5.17 mmol/L), her falsely decreased
serum sodium level of 121 mmol/L normalized to
139 mmol/L when measured by indirect potenti-
ometry (18).
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MINI-REVIEW
MITIGATION STRATEGIES
Several studies have compared the BGA and
PoC electrolyte analyzers with fully automated
chemistry analyzers for Naþ, potassium (Kþ), and
Cl estimation. Most authors have advocated the
use of a single technology for serial monitoring of
electrolytes for management of patients, or
recommended the use of dISE in critical care
cases (3, 20–22). However, several others have
suggested methods to correct the pseudo
hyponatremia.
Consideration of Serum Free Water Fraction
One such approach used the calculation of se-
rum free water fraction as initially suggested by
Waugh (23) and described in detail later by
Fortgens and Pillay (7). The Naþ measured by iISE
can be multiplied by the normal serum water
value of 93% and divided by its calculated value to
obtain an accurate estimate of serum Naþ.
However, in the formula
serum water % ¼ 99:1  ½1:1  10  5  lipid ðTGÞ concentration
in mmol=L – ð0:07  protein concentration in g=LÞ
only the TG concentration and total protein con-
centrations are considered. The cholesterol com-
ponent of serum lipids is conspicuously absent,
however, high cholesterol is recognized as a cause
of pseudohyponatremia although uncommon
(24).
Consideration of Osmolality
In a recent elaborate review on hyponatremia in
hematologic diseases, the authors discussed at
length the different hematological conditions that
are associated with pseudohyponatremia. These
conditions with underlying hyperglobulinemia,
hypertriglyceridemia, hypercholesterolemia, or
other causes of dyslipidemia mostly have isotonic
hyponatremia (serum osmolality being 280–
295 mOsm/kg) (25). Measurement of osmolality
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592 JALM | 589–595 | 07:02
Interference in Ion-Selective Electrodes
has thus been suggested as a tool to recognize
pseudohyponatremia. An observed difference be-
tween calculated and measured osmolality may
help the laboratory to identify suspected cases of
pseudohyponatremia. However, this would mean
requirement of additional cost, time, and
expertise.
Osm can be determined directly by measure-
ment of colligative properties of the solution,
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most commonly, using freezing point depression
or by measurement of vapor pressure. Since Naþ,
Cl, glucose, and urea are the major osmotic sub-
stances in normal plasma, osmolality can also be
calculated using the equation
mOsm=kg ¼ 1:86½Naþ ðmmol=LÞ þ glucose ½mmol=L
þ urea ðmmol=LÞ
Levels of sodium ion are required in the formula
for Osm, hence, erroneous measurement of so-
dium can cause erroneous calculation of Osm.
The same has been studied by looking at the ef-
fect of lipemia on calculated and measured Osm,
which revealed that lipemia does not affect Osm,
as measured by freezing point depression.
However, interference of lipemia with respect to
Naþ and Kþ concentrations measured using the
indirect ISE method causes low calculated Osm
values and may cause an erroneous increase in
the osmolal gap, especially in cases of toxic alco-
hol poisoning (26).
Calculation of Protein-Corrected Sodium
This problem of pseudohypernatremia
becomes all the more relevant in small children
who commonly have hypoalbuminemia; and
timely identification of hyponatremia is crucial for
proper management. Therefore, use of dISE has
been recommended by a couple of studies on
pseudohypernatremia or pseudonormonatremia
in sick infants (27, 28). In a study on 450 preterm
infants (weight <2500 g) admitted to the neonatal
intensive care unit, low plasma protein level
(<4.3 g/dL) was found to be an independent risk
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Interference in Ion-Selective Electrodes
factor for a Naþ level difference of >4 mmol/L be-
tween the two methods (27). However, since dISE
instruments mean additional cost to a hospital,
Stove et al. suggested using a protein-corrected
sodium result as an acceptable alternative (29). In
their study, they identified that for change of every
10 g/L in total protein concentration, a deviation
of 1.3 mmol/L is observed with Naþ indirect result.
They proposed the following equation:
protein  corrected Naþ indirect ¼ Naþ indirect  10:53
þ ð0:1316  total proteinÞ
On application of this equation on a prospective
cohort of 4006 patient samples the percentage of
conflicting data between iISE and dISE results sig-
nificantly decreased.
Another recent study, used the equation
fNaþ þ
adjusted ¼NaindirectISE [6.2  (0.16  albumin)]g
proposed by Story et al (12) to adjust serum so-
dium concentrations obtained from iISE for serum
albumin concentrations. They reported that the
differences between the direct and indirect meth-
ods became negligible after use of the equation
(30).
However, the use of corrective formulas, devel-
oped based on regression model-based algo-
rithms often seem impractical and tedious.
Besides, they do not hold true universally at all
ranges of measurement or for all interfering sub-
stance. Jones et al. derived regression equations
for the relationship between both the absolute
and relative difference between dISE and iISE and
the total protein concentration. But due to the
large spread of data around the regression line,
the authors disapproved the use of such equa-
tions and recommended dISE for electrolyte esti-
mation instead of iISE in routine clinical
laboratories (31).
Removal of Lipid Interference
Some studies suggest using either dISE or
methods such as ultracentrifugation or lipid clear-
ing agents (e.g., Lipoclear) for removal of
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MINI-REVIEW
interference due to lipemia for accurate estima-
tion of electrolytes in lipemic samples. Although
the interference was studied only in the case of
iISE previously, a study by Sen et al. (32) compared
two different dISE based equipment, namely
Vitros 250 (Raritan, New Jersey, USA) and HDC-
Lyte (Kolkata, India). In their experimental study,
Intralipid 10% (which is a sterile fat emulsion con-
taining soya oil, egg lecithin, and glycerol) was
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added in escalating concentrations to induce lipe-
mia in serum samples. Both the Naþ and Kþ val-
ues for the normal, hyper-, and hypoconditions
were found to have interference due to high TG
levels. The interference was observed beyond TG
level of 650 mg/dL (7.35 mmol/L) and was found
to be increasing as the TG levels rose to
1550 mg/dL (17.52 mmol/L) and beyond. The in-
terference was statistically significant, and the
maximum drift was seen with TG levels 1050 mg/
dL (11.86 mmol/L). They concluded that dISE, like
iISE at high TG concentrations, may influence elec-
trolyte measurements (32). However, the rationale
for the same has not yet been understood.
We have recently published a report with the in-
tention of studying the simultaneous effect of pro-
teins and lipids on electrolyte measurement by
iISE (3). Although the previously reported results
by different authors were corroborated, we could
not establish a combined formula for correcting
iISE results using protein, TG, and cholesterol.
CONCLUSION
It is important in clinical decision making to dis-
tinguish dyselectrolytemia from pseudodyselec-
trolytemia due to lipid or protein interference. A
correlation with clinical findings of dyselectrolyte-
mia is often therefore the first step to identify an
analytical error. However, laboratories need to be
proactive to prevent release of erroneous reports.
Mitigation strategies described by various
authors have been discussed here. In case
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MINI-REVIEW Interference in Ion-Selective Electrodes

discrepancy between clinical and laboratory find-
ings is found, the sample may be rerun on dISE.
However, dISE is not indicated in all situations. It is
necessary only when the sample is visibly lipemic,
or there is discrepancy between calculated and
measured Osm or, in cases with hyperglycemia. In
case instrument for measurement of dISE or Osm
is not available, formulas for calculation of cor-
rected sodium by free serum water or for protein-
both in large, automated laboratories and as BGA
or PoC devices because they provide rapid, inex-
pensive, accurate, and precise results. In spite of
high selectivity of the membranes that form an in-
tegral part of these electrodes, iISE are subject to
a lot of interference occurring due to abnormal
levels of proteins and lipids in serum. Therefore,

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corrected-sodium may be used. These formulas,
however, have not been validated on large num-
ber of samples and for their robustness (33).
Another option is removal of lipids from a lipemic
sample but it requires extra reagents or instru-
mentation. Use of all these strategies would ulti-
mately demand additional cost, time, and
expertise.
The ISEs play an indispensable role in clinical
chemistry testing for measurement of electrolytes
discrepancies arise between the dISE- and iISE-
based methods in these settings. Although several
attempts have been made to determine correc-
tive formulas, so far, no universally acceptable so-
lution to the problem has appeared. Hence, dISE
may be the safer bet in critical care settings and
interchangeable use of electrolyte results from dif-
ferent technologies should be avoided during se-
rial monitoring.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the follow-
ing 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of
data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be ac-
countable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are ap-
propriately investigated and resolved.

Conceptualization: S.K. Datta.

Writing, review & editing: P. Chopra, S.K. Datta.
Approval of final version: P. Chopra, S.K. Datta.
Both authors read and approved the final manuscript.
Authors’ Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

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