ISE Direct vs Indirect & iCa in clinical chemistry

Feb 2020

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 The opportunities of direct-
ISE based measurement of
ionized Calcium in Clinical
Chemistry
Stefan Köstler
Erba Technologies Austria GmbH
Feb 5th 2020

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Contents
• Introduction
• Direct and indirect ISE potentiometry
• Determination of free ionized and total
Calcium
• Perspectives for ionized Calcium
measurement

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Erba Technologies Austria
• One of several R&D sites of the Erba Mannheim
Group
• Located in Graz/ Austria / Europe

• Competence Center for

Developement of:
• Electrochemical and Optical Chemo- & Biosensors
• Smart (micro-)fluidic consumables & Cartridges
• Benchtop and POC instruments

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 Intro to ISE potentiometry
Na+

K+

Cl-

pH

Ca 2+
ISE – Membrane:
…
iphase boundary potential depends on
ion activity on solution (Nernst equation)

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Direct vs. indirect ISE methods
Direct ISE Indirect ISE
Principle: Electrode in direct Sample diluted (usually ≥1:10)
contact with undiluted before measurement.
sample
Results: Free ion concentration Total ion concentration
(activity) in the related to the whole sample
(extracellular) water volume
phase of the sample
Sample whole blood, serum, usually serum, urine
types: plasma, …
Analytes: Na+, K+, Cl-, Li+, pH, Na+, K+, Cl-, Li+ , Ca2+, …
Ca2+, Mg2+, …
Application: • Electrolyte & Blood • ClinChem Autoanalyzers,
Gas Analyzers (POC) • Urine electrolyte
• some Autoanalyzers measurement

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 Direct ISE Indirect ISE
Direct correlation to Reduction of electrode
physiologically relevant free ion interference:
concentrations • matrix effects
• Interfering substances
Broader spectrum of sample • liquid junction potentials
types (whole blood)
Prolonged electrode lifetimes
Less sample volume

Higher demands on ISE sensors Correlation to free ion conc. is

(dynamic range, selectivity, lost upon dilution
durability, …)
Erroneous results at abnormal
lipid or protein concentrations

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Electrolyte exclusion artifacts
• Electrolyte exclusion effect
(water displacement effect)
• Normal serum:
• 93 % water phase
• 7 % lipids, proteins
• Correction of indirect ISE data
for normal serum composition
J.B. Hall, et al., Principles of Critical Care, 4th Ed.

direct ISE 𝑁𝑁𝑁𝑁+ ok ok

𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝐻𝐻2 𝑂𝑂 𝑣𝑣𝑣𝑣𝑣𝑣.

indirect ISE 𝑁𝑁𝑁𝑁+ ok Na+ low

𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 𝑣𝑣𝑣𝑣𝑣𝑣.

•  erroneous results at abnormal lipid or protein conc. that

cause significant changes of the plasma water content.
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 Free ion concentrations
• In biological fluids a certain fraction of ions is usually bound or
complexed with proteins, organic or inorganic counterions, …
• ISE´s sense only free ions
• Strong dilution of samples
shifts the equilibria
 release of bound ions
•  Indirect ISE results are
close to the total ion conc.

Δctotal-free < 1% for several

common electrolytes
(Na+, K+, Cl-)
A.H.J. Maas, et al, Clin. Chem. 1985, 31, 482.
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 Ca 2 +

Ca2+ in the body

• Ca2+ homeostasis:
• intestine absorption
• exchange from bone
• reabsorption in kidney
• Free ionized Ca2+ (iCa2+)
is tightly regulated in
plasma.

• Total Calcium (tCa2+) in

plasma contains ca. 50%
of bound Ca2+ !
C. Higgins, 2007, acutecaretesting.org
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Total Calcium (tCa2+)
• Routinely determined:
Basic (Chem 8) or
comprehensive
metabolic panels
• Methods:
• Photometric assays
• Arsenazo III
• o-cresolphthalein
complexone (o-CPC)
• Indirect ISE
potentiometry
• AAS/AES (reference)

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Estimation of iCa from tCa
and albumin (protein)
• Majority of Ca2+ ions is bound to albumin
• Formula for adjustment of Ca values (e.g. Payne
1973): Caadj(mmol/L)= tCa(mmol/L)+0.02 [40–albumin(g/L)])
• iCa estimation from tCa is useable in healthy patients
with normal values of Albumin and lipids
• Many further variables: pH, total protein, Albumin-
to-globulin ratios, lipids, Mg, citrate
Calvi, et al, J.Am.Soc.Nephrol,2008

• Method used to derive albumin:

• Bromocresol Green BCG (original Payne equation)
• Bromocresol Purple BCP (mostly used today)

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 Multitude of equations
• Multiple reports on
erroneous results and
significant numbers of
wrong clinical
classifications

• Several authors
discourage the use of
albumin correction
formula at all.
They suggest using tCa
for screening and iCa for
further analysis

Mathau de-Antonio, Med Princ Pract,2016, 25, 219.

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Ionized (free) Calcium (iCa2+)
• Usually needs to be ordered separately
• Direct ISE method for serum or whole blood
• Ca2+ ionophores with good selectivity in polymer matrix
(e.g. ETH 1001)
• Developed as mature sensor technology since the 1980´s

• Integrated in modern POC blood gas

& benchtop electrolyte analysers

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When iCa2+ testing is required ?
• General:
• Abnormal serum protein composition
• Disturbance of acid/base balance
• Specifically:
• Critically ill patients
• Late stage CKD, renal failure
• Citrate anticoagulant in continuous hemofiltration
• Transfusions with citrated blood
• Hyperparathyroidism
• Hypercalcemia of malignancy
• Numerous requests that iCa2+ testing should
generally be preferred over tCa in clinical practice.
e.g.: P. Glendenigg, Ann.Clin.Biochem.2013; L.M. Calvi, et al, J.Am.Soc.Nephrol,2008, L. Larsson, et al, J.
Bone Mineral Res. 2003, etc., etc.

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Reasons for current prevalence
of tCa / Issues of iCa
• Lack of standardization/SRMs for iCa
• Equipment maintenance (electrode changes)
• Sample stability / Sample handling / Pre-analytics
• pH dependence
• anaerobic conditions
• timely analysis
• Anticoagulant interference
• EDTA, citrate
• Heparin
• High cost and effort of iCa tests due to separate manual
measurements
• no integration to CC auto-analysers so far
• workflow
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Standardization
• IFCC 2000/2 - Well defined Reference Method

• CLSI C39-A
•  NIST SRM 965a: assignment of iCa values

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pH dependence of iCa
• Binding of Ca2+ ions by proteins and other ligands is
pH dependent.
•  iCa2+ varies if sample pH changes before
measurement:
• Loss of CO2 to the atmosphere
• Lactate production via glycolysis
of cells

• pH dependence of iCa2+ is
well predictable in HCO3 buffer
system (in a range of ca. 7.2 – 7.6) McCudden, 2013, acutecaretesting.org

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pH adjustment of iCa
• a pH corrected iCa2+ value
(usually to pH 7.4)
can be calculated

• in cases of sample pH change

• opportunity of reporting
• actual iCa2+act.
• and pH corr. iCa2+pH7.4 values.

O. Müller-Plathe, Lexikon der Medizinischen Laboratoriums diagnostik, Springer

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Guidelines for sample
handling and pre-analytics
• Sample Handling/Pre-analytics
• IFCC (J. Autom. Chem. 1991)

• CLSI C31-A

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Suggested preanalytical
Procedures
• Whole blood:
• Measurement ideally within minutes (< 15 min) from
sampling
• chilling to 4°C if time till analysis > 30 min
• only Heparin anticoagulant (preferred dry of Ca
balanced) < 10 I.U.
• Serum:
• Anaerobic sample collection, complete filling of tube,
uncapping of tubes before measurement
• Centrifugation recommended within 30 min of sampling
and analysis within 30 min of centrifugation
• Haemolysis < 300 mg/dL

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Adoption of iCa testing and
preanalytical requirements
• In many centres iCa has been well adopted since its
introduction by clinicians for it´s superior relevance.

Bowers et al. 1986

• Sample handling and preanalytical challenges are

known and manageable
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Cost saving programs
• An iCa test might be up to 40x more expensive than tCa
calculating all manual labour costs in an US based clinic
(Ludwin 2014).
• Several hospitals have started programs to reduce iCa tests
due to high costs:
• tCa screening – iCa for further examination

G.S. Baird, Clin. Chem. 2009, 55, 533. Ludwin S, et al., J. Hospital Med. 2014; 9
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Erba Mannheim’s solution; aim
to increase availability iCa2+
• POC settings and small labs: EC90
• iCa is regularly included in the maintenance free
electrolyte sensor cartridge: No need to handle and
maintain separate Ca electrodes

• CC Auto-analysers for small – large Labs

• First direct ISE module for iCa in Auto-analysers
• Electrolyte sensor cartridge for maintenance free
operation

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Cartridge based direct ISE for
benchtop and auto-analysers

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Future integration of
direct ISE in NEXUS instruments

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