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brief-report2021
VDIXXX10.1177/10406387211001830Glucose method comparisons in Kemp’s ridley sea turtlesPerrault et al.

Brief Report

Journal of Veterinary Diagnostic Investigation

Comparison of 2 glucose analytical 2021, Vol. 33(3) 595­–599

© 2021 The Author(s)
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DOI: 10.1177/10406387211001830
https://doi.org/10.1177/10406387211001830
jvdi.sagepub.com
ridley sea turtles: dry chemistry of
plasma versus point-of-care glucometer
analysis of whole blood

Justin R. Perrault,1 Michael D. Arendt, Jeffrey A. Schwenter,

Julia L. Byrd, Kathryn A. Tuxbury, Nicole I. Stacy

Abstract. Blood glucose measurements provide important diagnostic information regarding stress, disease, and nutritional
status. Glucose analytical methodologies include dry chemistry analysis (DCA) of plasma and point-of-care (POC) glucometer
analysis of whole blood; however, these 2 methods differ in cost, required sample volume, and processing time. Because
POC glucometers use built-in equations based on features of mammalian blood to convert whole blood measurements to
plasma equivalent units, obtained glucose data must be compared and validated using gold-standard chemistry analytical
methodology in reptiles. For in-water, trawl-captured, immature Kemp’s ridley sea turtles (Lepidochelys kempii) from
Georgia, USA, we observed significant, positive agreement between the 2 glucose determination methods; however, the
glucometer overestimated glucose concentrations by 1.4 mmol/L on average in comparison to DCA and produced a wider
range of results. The discordance of these results suggests that POC glucometer glucose data should be interpreted in the
context of methodology- and brand-specific reference intervals along with concurrent packed cell volume data.

Key words: marine turtle; packed cell volume; plasma biochemistry; point-of-care device.

The use of point-of-care (POC) devices, including handheld
glucometers, is increasing in wildlife health studies as a
result of improved logistics and handling in the field, low
costs, small required sample volume, and near real-time
results.26,31 Glucose analysis in wild and rehabilitating sea
turtle patients provides important state-of-health informa-
tion, including acute stress, disease, various underlying met-
abolic derangements, and nutritional status; hence, glucose is
considered a critical diagnostic analyte and is important for
decision-making during sea turtle rehabilitation.24 Plasma or
serum glucose analysis by standard chemistry analyzers is
considered the gold standard compared to glucometers; how-
ever, laboratory chemistry analyses come at increased costs
with longer turnaround times.10,15 POC glucometers using
combined glucose oxidase and potentiometry have been
validated for use in 2 sea turtle species (green sea turtles,
Chelonia mydas; loggerhead turtles, Caretta caretta)16,22;
however, method comparison and validation have not yet
been reported in Kemp’s ridley sea turtles (syn. Atlantic rid-
ley; Lepidochelys kempii). Glucometers can be useful in field
assessments or during mass strandings of this critically
endangered species,30 especially as “cold-stunning” events
continue to occur during winter months along the East Coast
of the United States.11 Glucometer analysis of whole blood in
sea turtles has shown a slight positive bias in comparison to
plasma analysis by dry chemistry analysis (DCA).16,22 There-
fore, for free-ranging, immature Kemp’s ridley sea turtles
from Georgia, USA, our objectives were to 1) compare glu-
cose concentrations using 2 different analytical methods:
DCA using plasma and POC glucometer using whole blood;
2) determine the impact of packed cell volume (PCV) on glu-
cometer measurements; and 3) establish reference intervals
(RIs) for the glucometer.
Sample collection and processing followed previously
documented procedures.21 Briefly, Kemp’s ridley sea turtles
were captured using trawling3,4 and sampled from 31 May–
15 Jul 2016 (n = 16) and 5 Jun–19 Jul 2017 (n = 18) off
Loggerhead Marinelife Center, Juno Beach, FL (Perrault); Marine
Resources Division, South Carolina Department of Natural Resources,
Charleston, SC (Arendt, Schwenter); South Atlantic Fishery Management
Council, North Charleston, SC (Byrd); Animal Health Department, New
England Aquarium, Central Wharf, Boston, MA (Tuxbury); Aquatic,
Amphibian, and Reptile Pathology Program, Department of Comparative,
Diagnostic, and Population Medicine, College of Veterinary Medicine,
University of Florida, Gainesville, FL (Stacy).
1
Corresponding author: Justin R. Perrault, Loggerhead Marinelife Center,
14200 U.S. Highway One, Juno Beach, FL 33408. jperrault@marinelife.org
596
Perrault et al.

Table 1.  Measures of central tendency, range, and reference intervals (RIs; with 90% confidence intervals for upper and lower limits)
for plasma glucose using dry chemistry analysis (DCA) and whole blood glucose using a glucometer for in-water, immature Kemp’s ridley
sea turtles (Lepidochelys kempii). Parametric methods for sample sizes ≥ 20 but < 40 were used to calculate RIs.9 Normality was assessed
using the Shapiro–Wilk test; outliers were detected using the Reed test. All plasma samples were free of hemolysis and lipemia, except for
one sample with mild (1+) lipemia, which is not considered to cause interference using DCA.25

Mean ± SD Median Range n RI 90% LRL 90% URL

Glucose (plasma)*
 mmol/L 6.8 ± 1.1 6.7 4.7–9.2 34 4.7–8.9 4.1–5.2 8.4–9.4
 (mg/dL) (123 ± 20) (121) (85–166) (34) (85–160) (74–94) (151–169)
Glucose (whole blood)
 mmol/L 8.2 ± 2.2 8.0 4.9–13.5 33 4.0–12.5 2.9–5.1 11.4–13.6
 (mg/dL) (148 ± 40) (144) (88–243) (33) (72–225) (52–92) (205–245)
Data presented as SI units and conventional units (in parentheses). LRL = lower reference limit; SD = standard deviation; URL = upper reference limit.
* Data were previously reported,21 but are also included here for comparison to point-of-care glucometer concentrations.

Brunswick, GA. No animals were captured or sampled
more than once. Blood was collected from the external jug-
ular vein19 of each turtle and was placed into 10-mL sodium
heparin vacutainer tubes (Becton Dickinson). An aliquot of
well-mixed heparinized whole blood was used to determine
PCV and glucose using a POC glucometer (TRUEtrack;
Nipro Diagnostics) based on combined glucose oxidase and
potentiometry for use in whole blood.
Whole blood samples were centrifuged immediately
(within 5 min of collection) on the research vessel at 944 × g
(3,600 rpm) for 5  min. Plasma was separated, visually
assessed for the presence of lipemia and hemolysis,2,25 and
stored in liquid nitrogen onboard for several days until
stored in a shore-based ultralow freezer for up to 3 mo.
Frozen plasma samples were shipped overnight on dry ice to
the University of Miami Avian & Wildlife Laboratory for
glucose analysis using glucose oxidase and reflectance
photometry by dry slide chemistry analyzer (250XR; Ortho
Clinical Diagnostics).
Statistical analyses were performed using statistical
software (v.19.1; MedCalc). Agreement between glucose
results for the DCA using plasma and the POC glucometer
using whole blood was assessed using Passing–Bablok
regression and Bland–Altman difference plots. Differences
in glucose concentrations between the 2 methods were
determined using a paired sample t-test. Because the POC
glucometer works best at a PCV range of 0.30 to 0.55 L/L,
we also re-ran Passing–Bablok regressions and Bland–
Altman difference plots after removal of samples with a
PCV <  0.30 L/L (n = 8). Linear least-squares regressions
were used to establish a conversion equation between POC
glucometer concentrations and plasma glucose concentra-
tions. Lastly, RIs (95%, with associated 90% confidence
intervals) for whole blood glucose using the glucometer
were also calculated using parametric methods based on
recommendations for sample sizes ≥ 20, but < 40.9 Normal-
ity was assessed using the Shapiro–Wilk test; outliers were
detected using the Reed test.
We included 34 Kemp’s ridley sea turtles in our study.
The animals were considered to be in good health based
on 1) results of physical examination, 2) absence of exces-
sive epibiota, and 3) good visual body condition.21 Mean ± SD
glucose concentrations using the DCA with plasma were
6.8 ± 1.1 mmol/L (range: 4.7–9.2 mmol/L; n = 34) and using
the glucometer with whole blood were 8.2 ± 2.2 mmol/L
(range: 4.9–13.5 mmol/L; n = 33; Table 1). Mean ± SD PCV
was 0.32 ± 0.05 L/L (range: 0.20–0.44 L/L; n = 33; Table 1).21
One adult female (min. straight carapace length = 61.5 cm)
was excluded from the calculation of RIs, because this indi-
vidual was the only mature turtle captured (breeding size of
Kemp’s ridleys is ≥ 58 cm standard straight carapace length)8
and RIs established here are for immature Kemp’s ridley sea
turtles; a second turtle was missing a reading for glucose by
glucometer.
A paired sample t-test indicated that glucose measure-
ments differed significantly (t(32) = 5.577; p < 0.001)
between glucose concentrations determined by DCA and the
POC device (Fig. 1A). Passing–Bablok regression analysis
showed a strong linear association between the 2 methods
(rs = 0.72; p < 0.001; Fig. 1B); however, both systematic and
proportional differences were observed between the 2 meth-
ods, and the Bland–Altman plot indicated a negative bias
(−1.4 mmol/L; limits of agreement: −4.4 and 1.5 mmol/L),
with the POC glucometer giving higher results than the DCA
(Fig. 1C). A total of 29 of 33 (88%) samples had higher glu-
cose concentrations when analyzed by glucometer compared
to the DCA. When glucose measurements associated with
PCV values < 0.30 L/L were removed from method compari-
sons, the Passing–Bablok regression rs value increased (0.72
to 0.80) and the Bland–Altman bias decreased (−1.4 mmol/L
to −1.2 mmol/L); however, both systematic and proportional
differences were still observed between the 2 methods.
Previous studies on glucose comparisons using DCA
and POC glucometers have been conducted in green and
loggerhead sea turtles. 16,22 The glucometer brand that we
used is different from the one employed in those studies16,22;
 Glucose method comparisons in Kemp’s ridley sea turtles
Figure 1.  A. In a paired sample t-test, blood glucose
concentrations of Kemp’s ridley sea turtles (Lepidochelys kempii)
from Georgia, USA, significantly differed when using a dry
chemistry analyzer (DCA; Vitros 250; Ortho-Clinical Diagnostics)
compared to a point-of-care (POC) glucometer (TRUEtrack;
Nipro Diagnostics). The central box represents the lower-to-upper
quartile (25th–75th percentile), with the middle line representing
the median. The vertical lines extend from the minimum to the
maximum values. No outliers were present. Asterisks above the box
indicate a significant difference at p < 0.001. B. Passing–Bablok
Figure 1.  (continued)
597
(continued)
regression comparing glucose in plasma using a DCA and in whole
blood using a POC. The dashed gray line indicates the line of
identity (y = x); the solid black line is the line of best fit. The dashed
black lines are the 95% confidence intervals (CIs) of the slope.
C. Bland–Altman difference plots comparing glucose in plasma
using a DCA and in whole blood using a POC. The dashed gray
line represents the line of identity, the solid black line indicates the
mean difference between the 2 methods, and the black hatched lines
are the 95% limits of agreement. An intercept close to 0, a slope
close to 1, and a bias close to 0 indicate the fewest systematic and
proportional differences between methodologies. These 2 methods
had a strong linear association (rs = 0.72), but there was a bias of
1.4 mmol/L between the 2 methods.
however, both brands use the same electrochemical-based
methodology with glucose oxidase and potentiometry ver-
sus other optical methodology-based glucometers. Glucose
consumption by RBCs continues after blood collection, and
concentrations have been shown to rapidly decrease with
delayed plasma separation times and sample exposure to
increased temperatures. 7,16 Because our samples were
processed immediately (i.e., within 5 min of collection),
we can assume that our glucose measurements, both by
DCA and glucometer, were not affected by continued RBC
consumption.
Baseline plasma glucose concentrations for Kemp’s rid-
ley sea turtles range from 2.3 to 11.5 mmol/L.1,5,6,14,17,24,25
Results from the DCA (range: 4.7–9.2 mmol/L) showed clin-
ically normal glucose measurements for all immature turtles
sampled. Similarly, glucose results from the glucometer
(range: 4.9–13.5 mmol/L) also fell within the normal range
of glucose concentrations for Kemp’s ridley sea turtles,
except for 2 samples (12.0 mmol/L and 13.5 mmol/L). The
effects of capture technique cannot be ruled out, given that
forced submergence, albeit minimal, may have impacted
blood glucose concentrations.21
Similar to other studies, the POC glucometer produced
significantly higher concentrations (by 1.4 mmol/L in our
study) compared to DCA results.16,22,27,31 The 2 methods
were strongly correlated; however, 25 of 33 (76%) and 8 of
33 (24%) samples had a ≥ 10% and ≥ 40% difference
between the methodologies, respectively. Hence, this brand
of glucometer consistently produced results that were
above the total allowable error of 10% for glucose set by
the American Society for Veterinary Clinical Pathology,13
which raises concern that this bias may affect clinical deci-
sions. In green and loggerhead sea turtles, POC glucome-
ters overestimated glucose concentrations by 0.1 mmol/L
and 0.4 mmol/L, respectively.16,22 This demonstrates the
need for analyzer- and brand-specific RIs for glucometers,
despite the use of identical analytical methodology by dif-
ferent manufacturers.
Handheld glucometers are designed for optimal use in
humans, and reasons for the observed variations and range in
results of our study likely include dissimilarities in analytical
598
Perrault et al.
methodologies (DCA uses hexokinase and glucose-
6-phosphate dehydrogenase; the POC glucometer uses glu-
cose oxidase and potentiometry), reptilian RBC morphology
(e.g., nucleated RBCs in reptiles vs. non-nucleated RBCs in
mammals; variations in nuclear and cell size during phases
of erythroid regenerative responses), PCV, electrolyte con-
centrations of the samples of interest, in addition to a vari-
ety of pre-analytical factors (e.g., prandial status, filling of
glucose strips, partial pressure of oxygen, and even drying
of isopropyl alcohol on skin before venipuncture).10,16,22
These observed differences are also important from a clin-
ical perspective. If using a glucometer to determine glu-
cose concentrations in rehabilitating sea turtle patients or
during population health assessments, caution is warranted
in the interpretation of data because of consistently higher
glucose data by glucometer across studies compared to
regular chemistry analysis.16,22 For example, hypoglyce-
mia may go unnoticed or may be more severe than realized
if using only a glucometer to determine glucose concentra-
tions. These methodological differences should always be
considered, given that decisions for treatment (e.g., fluid
administration) based on these results may differ depend-
ing on which method and brand were used. Linear regres-
sion analysis revealed a significant relationship (r2 = 0.61;
p < 0.001) between whole blood POC glucose concentra-
tions and plasma DCA glucose concentrations, which can
be described as:
Glucose DCA = ( 0.39 × Glucose POC ) + 3.58
Using this equation, the coefficients of variations between
the actual plasma DCA glucose concentrations and the calcu-
lated concentrations ranged from 0.1% to 11.3% (mean ± SD:
3.7 ± 3.2%), indicating that plasma glucose concentrations
can be accurately predicted by whole blood glucometer con-
centrations using the TRUEtrack glucometer in Kemp’s rid-
ley sea turtles.
It is possible that the reduced accuracy of the glucose
measurements in sea turtles using human glucometers occurs
as a consequence of the comparatively low PCV of sea tur-
tles compared to humans, given that the device is most accu-
rate at PCVs of 0.30 to 0.55 L/L.16,27 PCV has been shown to
influence POC glucose measurements in humans27 and
domestic mammals.12,18,20,23,28,29 These units also use built-in
equations specific for mammalian blood to convert whole
blood measurements to plasma equivalent units, and such
equations may not be suitable for use with reptiles.10,16 With
PCV < 0.30 L/L or at the lower range of PCV for the species,
POC glucose readings in whole blood tend to be higher than
with mid- or upper-range PCV. Hence, an anemic sea turtle
patient with PCV < 20% will have higher POC glucose, pos-
sibly masking underlying hypoglycemia. Such limitations of
using glucometers must be considered in decisions regarding
additional testing and medical care.
Acknowledgments
We thank the crew of the research vessel Georgia Bulldog, includ-
ing Lindsey Parker, Lisa Gentit, and H. “Truck” McIver. We also
thank the team involved with blood processing: Christopher Evans,
Austin Pickhardt, Julie Dingle, Sharleen Johnson, S. Michelle Pate,
and Shannon Howard. We thank Drs. Craig Harms and Charlie
Innis for providing feedback on earlier drafts of this manuscript.
Declaration of conflicting interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
Funding
This project was supported by the National Marine Fisheries Service
Southeastern Regional Office Award NA13NMF4720182.
Compliance with ethical standards
Our study was carried out in accordance with Endangered Species
Act Section 10(a)(1)(A) permit 19621, Georgia Department of Nat-
ural Resources Scientific Collection Permit CN21303, and approved
Institutional Animal Care and Use Committee protocol UF IACUC
201706823.
ORCID iD
Justin R. Perrault https://orcid.org/0000-0002-5046-6701
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