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Evaluation of agreement between a laboratory- based and a field-based blood analyser for analysis of selected biochemical analytes in farmed Atlantic salmon (Salmo salar L.)

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Agreement between a point of care device and a laboratory blood analysis machine for parameters lactate, sodium, potassium and chloride was evaluated using samples from two different Atlantic salmon populations. The results were summarised using scatter plots and calculations of Lin´s concordance correlation coefficient, and Pearson´s correlation coefficient. Good concordance and correlation were found for lactate, while the concordance and correlations for the blood ions were very low.
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Bull. Eur. Ass. Fish Pathol., 41(1) 2021, 17
* Corresponding author’s email: liv.ostevik@shvetgroup.com
Evaluation of agreement between a laboratory-
based and a eld-based blood analyser for
analysis of selected biochemical analytes in
farmed Atlantic salmon (Salmo salar L.)
L. Østevik1*, M. Kraugerud1, H. Rodger2,
A. Nødtvedt3, M. Alarcón1
1 Fish Vet Group Norge AS, Hosveien 21-23, 0275 Oslo, Norway; 2VAI Consulting,
Kinvara, Co. Galway, Ireland; 3Department of Production Animal Clinical Sciences, Faculty
of Veterinary Medicine, Norwegian University of Life Sciences, Oslo Norway
Abstract
Agreement between a point of care device and a laboratory blood analysis machine for parameters
lactate, sodium, potassium and chloride was evaluated using samples from two dierent Atlantic
salmon populations. The results were summarised using scaer plots and calculations of Lin´s
concordance correlation coecient, and Pearson´s correlation coecient. Good concordance and
correlation were found for lactate, while the concordance and correlations for the blood ions were
very low.
Introduction
Clinical biochemistry is a well-established
diagnostic tool in both human medicine and
veterinary medicine of terrestrial animals.
Serum or plasma from a blood sample can be
analysed for panels of indicators of organ func-
tion. Typically, the technique involves measure-
ment of an enzyme, a metabolic product or an
ion. The combined information from the panel
of analytes can give diagnostic and prognos-
tic information and can be used to assess the
progression of a disease and assist in making
decisions about treatment and management
(Thrall, 2012). In salmonids, gill diseases such
as amoebic gill disease (AGD) and tenacibacu-
losis have been shown to aect plasma levels
of chloride, sodium, potassium, cortisol and
lactate (Hvas et al., 2017) and lactate (Powell et
al., 2005), respectively. Increased lactate levels
were found in salmon with infectious salmon
anemia (Olsen et al., 1992) and in sh with
myopathies such as in pancreas disease (PD)
(Rodger et al., 1991). Lactate is also used as an
indicator of stress during handling (Iversen et
al., 2005). The application of clinical chemistry
in clinical sh medicine is still relatively limited
(Braceland et al., 2017), however, availability
of blood analysis devices that are easy to use
and deliver results rapidly may contribute to
changing this.
Handheld and portable devices for “point-of-
care” (POC) analysis have been developed for
clinical chemistry in human and veterinary
18, Bull. Eur. Ass. Fish Pathol., 41(1) 2021
medicine. Such devices have the potential to be
used at the sh farm, yielding rapid informa-
tion about sh health without the delay caused
by sending samples for laboratory analysis.
However, there is limited knowledge about
the practical use and reliability of such POC-
devices in Atlantic salmon. The iSTAT (Abaxis
Europe GmbH) is a POC-device used in human
and small animal veterinary medicine which
has the advantage of being able to analyse a
wide range of blood analytes, including ions,
enzymes and gases. It is easy to use, and the
small size means it can easily be transported
to sh farms or hatcheries for on-site analysis.
The aim of this study was to test the agreement
between the point of care device iSTAT in the
eld and a laboratory blood analysis machine
for the parameter’s lactate, sodium, potassium
and chloride.
Materials and Methods
Blood samples were collected at two dier-
ent occasions from two dierent farmed sh
populations in Norway. First, 21 Atlantic salmon
post-smolts weighing approximately 200 g
kept in seawater with a temperature of 10°C
at NIVA Research Facility at Solbergstrand
were sampled. Four sh had skin ulcers and
a proportion of the sh were below normal
body condition or had erosions of the n tips.
Fish were intended for use in another research
project and sh selected for sampling came from
3 dierent tanks. Euthanasia was achieved with
an overdose of benzocaine (Benzoak vet, ACD
Pharmaceuticals AS, Leknes, Norway). Blood
was subsequently collected from the caudal
vein (21 sh) and additional blood was drawn
from the heart (2 sh), directly into lithium-
heparinised tubes (Vacuee, Greiner Bio-One
GmbH, Kremsmünster, Austria).
The second sampling was performed at a sea site
in Møre og Romsdal (north western Norway)
where grossly visible gill lesions and a history
of gill disease had been recently reported. At
the time of the sampling the 41 sampled sh
weighed from 2.4 to 4.9 kg and no clinical signs
of disease or elevated mortality rates were
evident. The sea temperature was measured
at 5.9 and 6°C on the two sampling days. Fish
were sedated with benzocaine (Benzoak vet,
ACD Pharmaceuticals AS) as per producer
recommendations for lice counting (sea lice,
Lepeophtheirus salmonis). After lice counting sh
were placed in an approximately 1000 L salt-
water tank and sedated with isoeugenol (Aqui
-S vet. Scan Aqua, Årnes, Norway) at 5 mL per
cubic meter water and subsequently euthanised
with an overdose of metacaine (Finquel vet.
Scan Aqua) in an anesthetic bath with an ap-
proximate concentration of 200 mg/L. Fish were
euthanised one at a time and were considered
dead when no gill movement was observed in
the anesthetic bath. Euthanasia was immedi-
ately followed by drawing of blood from the
caudal vein directly into lithium-heparinised
tubes (S-Monovee LH, Sarsted, Germany).
Approximately 120 or 250 μL of whole blood
(120 μL per cartridge) was used for iSTAT anal-
ysis. The remaining heparinised blood was
cooled and centrifuged in batches at 5000 rpm
for 10 min. Plasma was transferred into 2 mL
Eppendorf tubes and either chilled until arrival
at the lab and subsequently frozen (sampling
1) or stored at -20oC until shipping to the Fish
Vet Group lab on ice (sampling 2).
At sampling 1 iSTAT analysis was performed
as soon as possible after sampling, however,
time from sampling to analysis varied from
Bull. Eur. Ass. Fish Pathol., 41(1) 2021, 19
a few mins to approximately 1.5 h. The delay
between sampling and analysis is likely to have
led to an increased temperature of the aected
samples. The analyser was acclimatised to the
ambient temperature for at least 30 min prior
to the rst analysis and all analysis was per-
formed at room temperature (approximately
20-22°C). Each blood sample was analysed with
two separate cartridges; CHEM8 and CG4+
(Abaxis Europe GmbH; Griesheim, Germany)
on a VetScan iSTAT1 handheld blood analyser
(Abaxis Europe GmbH; Griesheim, Germany).
Parameters measured included sodium (Na),
potassium (K), chloride (Cl), hematocrit (Hct)
for CHEM8 and lactate for CG4+. For sampling
2, iSTAT analysis was performed within 5 min
after sampling. Samples analysis was performed
in the boat cabin, and ambient temperature
varied throughout the two days of analysis es-
timated at approximately 13-15°C and increas-
ing to 20-25°C as the cabin was heated by the
engine and the sun. Each sample was analysed
using a single cartridge (CHEM8). The iSTAT
measures sodium, chloride and potassium by
direct ion-selective-electrode (ISE) potentiom-
etry. Hematocrit and lactate are measured in-
directly by whole-blood conductometry and
by amperometry of hydrogen peroxide after
conversion of lactate to hydrogen peroxide
and pyruvate by lactate oxidase, respectively.
Laboratory blood analysis was performed
on plasma (4°C) after thawing of previously
frozen samples. Analyses were performed for
sodium, potassium, chloride, urea and lactate
using ABX Pentra kits (Horiba Medical, Irvine,
CA) on the ABX Pentra C400 (Horiba Medical,
Irvine, CA) according to instrument protocols.
In this device, ions are measured by direct ion-
selective-electrode (ISE) potentiometry, while
lactate was measured using spectrophotometry
(colorimetry) with lactate oxidase/peroxidase
as reagent.
Statistical analysis of results was undertaken
in STATA (StataCorp. 2015. Stata Statistical
Software: Release 14. College Station, TX: Stata-
Corp LP.). Scaerplots were produced for each
parameter to provide a visual presentation of
iSTAT versus Pentra values. Agreement was an-
alysed per sampling event and across sampling
events, including all samples. Direct agreement
(concordance) between the laboratory-based
machine and the POC-device was assessed by
the concordance correlation coecient (Lin,
1989). Correlation between the two readings
were also assessed by Pearson’s correlation
coecient. Both correlation coecients can
range between 1 and -1, with 0 indicating no
agreement. The concordance correlation coef-
cient more directly compares two sets of test
results compared to the Pearson’s correlation
coecient which ignores the scales of the test
sets. A concordance correlation coecient of
1 means perfect agreement between two test
sets, while a Pearson’s correlation coecient of
1 and -1 means total positive or negative linear
correlation, respectively (Dohoo et al., 2014).
Results
Results of blood and plasma analysis are sum-
marised in Table 1. The iSTAT gave no valid
readings from a relatively high proportion
of the samples, 19/62 (31%) and10/62 (16%)
for potassium and chloride, respectively. Ad-
ditionally, a large fraction of readings were
below the highest or lowest detectable levels for
both ions, leaving only 10 duplicate measure-
ments for chloride and 33 duplicates for potas-
sium. In contrast all samples analysed with the
20, Bull. Eur. Ass. Fish Pathol., 41(1) 2021
Pentra resulted in presumed valid readings
for all analytes. Both the correlation and
concordance correlation coefficient (Table
2) and scatter plot indicated poor agreement
between the two devices for chloride (data
not shown), sodium (Figure 1) and potas-
sium (Figure 2).
The iSTAT lactate measurements ranged from
0.69 to 2.42 mmol/L. Plasma lactate meas-
ured by the Pentra ranged from 1.03 to 3.47
mmol/L. Using the conversion method re-
ported by Gallagher et al. (2010) whole blood
equivalents (converted lactate values) were
generated by multiplying plasma lactate
values by 1 – hematocrit. The scatterplots
(Figure 3 and 4) and Pearson correlation
coefficient shows a high correlation between
devices for both converted and unconverted
lactate measurements, while concordance
is high only between iSTAT and converted
Pentra lactate values (Table 2).
Na
mmol/L
(Lab)
Na
mmol/L
(POC)
K
mmol/L
(Lab)
K
mmol/L
(POC)
Cl
mmol/L
(Lab)
Cl
mmol/L
(POC)
Lactate
mmol/L
(Lab)
Lactate
mmol/L
(Lab
con†)
Lactate
mmol/L
(POC)
Sampling
1
155.7
(17.8)
n = 20
156.5
(8.78)
n = 21
1.78
(1.29)
n = 20
2.0
(0.4)
n = 3
138.8
(16.6)
n = 20
130.7
(6.1)
n = 11
2.36
(0.76)
n = 20
1.66
(0.50)
n = 20
1.57
(0.51)
n =21
Sampling
2
167.7
(11.5)
n = 41
166.4
(6.0)
n = 31
1.70
(1.25)
n = 41
3.2
(0.8)
n = 31
141.6
(9.6)
n = 41
-
n = 0 ND ND ND
Tabl e 1 . Mean values for analytes as measured by the POC-device and laboratory analyser. Standard
deviation is given in parentheses. Mean values are calculated for numerical values only. Calculation of
mean values was not possible for the analyte chloride for one or both samplings due to no and few samples
with numerical values. ND = not done. †Converted values generated by multiplying original values with a
conversion factor C (C = 1– hematocrit/PCV).
Na mmol/L K mmol/L Cl mmol/L Lactate mmol/L Lactate
converted
mmol/L
Sampling
1
0.16
(0.23)
n =20
- - 0.43
(- 0.52)
n= 10
0.49
(0.95)
n = 20
0.89
(0.91)
n = 20
Sampling
2
0.43
(0.47)
n = 31
0.09
(0.30)
n = 30
- - -
Over all
samples
0.38
(0.44)
n = 51
-0.03
(-0.07)
n = 33
- 0.52
(- 0.43)
n = 10
0.49
(0.95)
n = 20
0.89
(0.91)
n = 20
Tabl e 2 . Agreement between the POC-device and laboratory analyser. For each analyte concordance
correlation coecient and Pearson’s correlation coecient (in parentheses) and number of observations
are listed.
Bull. Eur. Ass. Fish Pathol., 41(1) 2021, 21
Discussion
Our results indicate that the Vetscan iSTAT
POC-device does not perform well for analysis
of the electrolytes chloride or potassium in the
blood of Atlantic salmon in saltwater. A large
number of samples were missing or below the
highest or lowest detectable levels for these pa-
rameters, and the seings for upper and lower
limits of detection for several analytes seems to
be unsuited for the blood concentrations found
in Atlantic salmon. There was a good agreement
between the iSTAT and the converted Pentra
C400 values for lactate, though values from
the POC-device cannot be directly compared
to plasma values from the laboratory analyser
because they use dierent source materials
(whole blood versus plasma).
The iSTAT measures lactate levels in whole
blood as opposed to plasma and this likely ex-
Figure 1. Scaerplot of sodium levels as measured by iSTAT and Pentra. One outlier sample excluded for
beer visualisation
Figure 2. Scaerplot of potassium levels as measured by iSTAT and Pentra
22, Bull. Eur. Ass. Fish Pathol., 41(1) 2021
plains the higher concentrations of lactate meas-
ured by the laboratory analyser (Goodwin et al.,
2007). A lower lactate concentration is found in
the extracellular fraction of whole blood if ana-
lysers use a volume dependent measurement
method due to the dilution eect of erythro-
cytes in the sample. Using a conversion method
for plasma lactate substituting hematocrit for
packed cell volume (Gallagher et al., 2010) the
agreement between lactate values measured
between the two devices was good (concordance
correlation coecient 0.893), but not excellent
as reported by the said authors. However, this
conversion requires that packed cell volume or
Figure 3. Scaerplot of lactate levels as measured by iSTAT and Pentra. (Data from pilot 1 only).
Figure 4. Scaerplot of lactate levels as measured by iSTAT and converted lactate levels measured by Pentra.
(Data from pilot 1 only).
Bull. Eur. Ass. Fish Pathol., 41(1) 2021, 23
hematocrit measurements are also available. In
our case hematocrit was measured by iSTAT
which does not directly measure packed cell
volume, and previous research (Borissov et
al., 2019; Harter et al., 2014) has shown that
hematocrit in sh is not accurately measured
by the iSTAT when compared to conventional
laboratory methods. The use of centrifugation
for assessing packed cell volume and using
these values for generation of the converted
plasma lactate values might have improved
the concordance between the two devices. In
addition, both the plasma and whole blood
concentrations of lactate in some samples in this
study are likely falsely elevated due to variable
delay in sample analysis during sampling 1.
In humans lactate levels will increase rapidly
if samples are not immediately analysed or
frozen (Andersen et al., 2003), but in our case the
time prior to whole blood analysis and plasma
separation and freezing was almost identical,
thus this problem has likely not inuenced the
agreement analysis to a large extent.
The iSTAT measured analyte levels in whole
blood immediately after sampling while frozen
and thawed plasma was used for Pentra C400.
These dierences in pre-analytic treatment of
the samples may lead to real dierences in
analyte levels (Braceland et al., 2017; Braun et
al., 2015). In fact, Braceland et al. (2017) showed
small but statistically signicant dierences in
sodium, potassium and chloride concentrations
in duplicate samples of serum and plasma from
Atlantic salmon. A minimal eect of several
freeze thaw cycles was also found. While sig-
nicant dierences are found when measuring
lactate levels in whole blood versus plasma, this
eect in not so clear for the ions sodium, chlo-
ride and potassium. A study comparing whole
blood and plasma as sources for measurement
of sodium in humans showed good agreement
between measurements, but a positive bias of
iSTAT measurements when analysing whole
blood from hyponatremic patients (Geoghegan
et al., 2015). Two studies with human patients
using paired samples and comparing POC-
devices measuring analyte levels in whole blood
to a central laboratory device measuring analyte
levels in plasma found a small mean dierence
for sodium, potassium and chloride levels (King
and Campbell, 2000). However, a signicant
dierence was found in individual sodium,
chloride, and potassium levels measured by the
two devices, and a signicant dierence was still
evident when plasma instead of whole blood
was used as a sample source for the POC-device
(Morimatsu et al., 2003).
The Pearson correlation coecient ignores the
scales of the two sets of results being compared,
while the concordance correlation coecient
does not (Dohoo et al., 2014). The concordance
correlation coecient beer reects the level of
agreement between two sets of test results and is
preferred for comparing such results, however
it is sensitive to dierences between the sample
materials or the handling of samples as in this
study. However, any dierence caused by the
analysis of plasma versus whole blood, storage
and freezing of samples would likely cause a
deviation in the same direction, i.e. analytes
should be systematically increased or decreased
in one sample set compared to the other. A
high correlation between the values generated
by the two analysers would then be expected
if both analysers were performing well. Thus,
even if the use of plasma and whole blood and
dierences in pre-analytical treatment of the
samples could partially account for the low
24, Bull. Eur. Ass. Fish Pathol., 41(1) 2021
agreement between the two analysers in our
study, dierences in sodium, potassium and
chloride are also likely due to real dierences
in measurements performed by these devices.
The iSTAT is portable and easy to use, however
the device is sensitive to ambient temperature
which can aect the analysis (pers. comm.
Elizabeth Norstrøm, Kruuse AS) and requires
time to acclimatise onsite. This makes the use
of the device less practical in clinical practice
as time for farm visits may be limited and a
space suitable for blood analysis with stable
temperature could be dicult to nd. During
the rst day of the second sampling at the sea
farm the temperature in the room where iSTAT
analysis was performed was rising rapidly due
to sunlight. This is a possible explanation for
the device not producing any values for potas-
sium and chloride for seven sh (sampling 2,
sample 13-19), while all results for sodium for
these sh were above the upper limit of 180
mmol/L that day. These potential problems
with the device do not change the results of
the statistical analysis as the missing values
were not included in nal analysis. The use of
a room with stable temperature and improved
standardisation of sampling and analysis could
potentially have strengthened the test results
obtained from the iSTAT, however the aim of
this study was to test the applicability of the
iSTAT in clinical practice which often oers
less than ideal conditions.
Our results indicate that the iSTAT is not a reli-
able tool for assessment of the selected ions
in Atlantic salmon and are in line with earlier
results in other sh species. Stoot et al. ( 2014) re-
viewed studies using or validating POC-devices
for use in sh, birds and mammals. Previous
work performed on rainbow trout, Oncorhyn-
chus mykiss, by Harter et al. (2014) aempted to
validate the iSTAT system for use in this species.
In this study CG8+ cartridges were used and
iSTAT was found to give meaningful results
for pH, while the values were not meaningful
for hematocrit or parameters sodium, partial
pressure of carbon dioxide, partial pressure
of oxygen or bicarbonate. Similarly, a study in
cod (Gadus morhua) comparing the iSTAT with
conventional laboratory techniques found that
the POC-device was inaccurate for measure-
ment of pH, pO
2,
hematocrit, sodium, potassium,
calcium and hemoglobin (Borissov et al., 2019).
A validation study in Seminole killish (Fundu-
lus seminolis) (DiMaggio et al., 2010) found that
iSTAT could not be used to assess hematocrit
or plasma ions in this species. Harrenstien et
al. (2005) aempted to validate the iSTAT for
two rocksh species (Sebastes melanops and Se-
bastes mystinus), but found signicant dierences
between measurements performed with the
iSTAT and conventional laboratory methods.
These dierences have been related to the fact
that the iSTAT and its algorithms and constants
were developed for use in humans (Harter et al.,
2014). Dierences in red blood cell morphology
and blood temperature between humans and
sh, as well as other physiological dierences,
are likely to explain erroneous measurements in
sh. An eect of dierent blood temperatures,
hematocrit and/or PCO
2
on measurement of ions
was also found and based on this the authors did
not recommend the use of conversion factors to
correct these iSTAT-measurements (Borissov et
al., 2019; Harter et al., 2014).
Acknowledgements
The authors would like to thank Øystein
Evensen for help with sampling during sam-
Bull. Eur. Ass. Fish Pathol., 41(1) 2021, 25
pling 1, Elizabeth Norstrøm and Kruuse AS for
allowing us to borrow the iSTAT, Tanja Hogstad
for plasma analyses at FVG and Mowi sta
for help and support during sampling 2. This
work was supported by the Norwegian Seafood
Research Fund (project number 901515), Mowi
ASA and Fish Vet Group AS.
The authors LØ; MA, MK work at Fish Vet
Group Norge AS, a commercial diagnostic labo-
ratory that oers clinical chemistry as a service
to the aquaculture industry.
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