Immediate on-site glucose measurements in dogs
– refinement of procedure
Valeria Golozoubova, Jes Tovborg Jensen, Jeanet Løgsted Nielsen and Andrew Makin
CiToxLAB Scantox, Hestehavevej 36A, Ejby, LilleSkensved, 4623, Denmark
Introduction
Glucose measurement in a small blood sample analysed without a delay provides clear benefits
in the studies where blood glucose levels are expected to be reduced (e.g. tests of insulin) both
practically and ethically (immediate actions without a delay can be taken in cases when hypoglycaemia is suspected). One Touch Ultra glucometers are used successfully in humans but are neither
calibrated nor tested for use in animals. The dog has been selected as the test model in the present
study because dog is the most commonly used animal model in the types of studies where direct
glucose measurement would be directly beneficial.
The objective of this study was to validate the One Touch Ultra glucometer for glucose measurements in dog blood, with special focus on low blood glucose concentrations (identification of
hypoglycaemia). In addition, direct comparison of glucose measurements in serum and in EDTAstabilised plasma was performed.
glucose level of 1.7 mmol/L. Values below the lower limit of detection are displayed on the screen of
One Touch Ultra as “LO”. As the corresponding values in dogs are regarded as extremely low and are
often associated with severe clinical signs where immediate actions for counteraction of hypoglycaemia (food or glucose p.o or i.v., dependent of the state of the animal) have to be taken. One Touch
Ultra values between 1.1 and 1.9 mmol/L (corresponding to plasma glucose levels of 2.1-3.0 mmol/L),
are regarded as very low. Thus, the low limit of detection of glucometer is sufficient for identification
of a hypoglycaemic state requiring intervention in dogs. Table 1 below gives the overview of values
obtained using glucometer corresponding to plasma glucose values in dogs.
Table 1: Evaluation of glycemic status of dogs
One Touch Ultra, mmol/L
Materials and methods
Animals and treatment. The study was performed in grown beagle dogs of the strain HsdRcc:DOBE.
Comparison of glucose measurements in whole blood and in plasma/serum was performed using
multiple sampling in 3 male dogs. Comparison of glucose measurements at different sites (foreleg,
throat and ear) was performed using multiple sampling in four dogs (2 males, 2 females). In order
to reliably validate the method across a broad range of glucose levels (as broad as possible without
jeopardising the health of the animals), blood sampling was performed in before (normoglycemic
animals) and after (hypoglycaemic animals) treatment with insulin analogue (at time points where
the glucose-lowering effect was most prominent). The range of measured plasma glucose levels in
the study was between 1.7 – 6.5 mmol/L.
Glucometers. Two models of One Touch Ultra glucometers from the same producer (LifeScan, division
of Cilag GmbH International) were used in the study, OneTouch® UltraEasy® and OneTouch® Ultra®2.
Both models use the same type of test strips (OneTouch® Ultra® Test Strips), and are controlled with
the same control solution (OneTouch® Ultra® Control Solution).
Blood samples. For direct measurements using One Touch Ultra glucometers, only one drop of
full blood was required, and the sample was measured instantly. For measurements of glucose in
plasma and serum, blood samples of approximately 3 mL were drawn from the jugular vein. The
blood was sampled into two types of vacutainers – plain (serum) and containing EDTA as anticoagulant (plasma). For plasma samples, the blood was placed in ice water for a maximum of 30 minutes
before centrifugation. Plasma was separated by centrifugation (10 min, 1500 G, +4°C) and frozen at
-18°C until analysis. For serum samples, the blood was placed at room temperature for a maximum of
1 hour before centrifugation. Serum was separated by centrifugation (10 min, 2000 G, RT) and frozen
at -18°C until analysis. Plasma and serum glucose analysis was performed using an Hitachi 917.
Results
Comparison of glucose measurements in whole blood and in plasma/serum
The reliability and quality of measurements using One Touch Ultra in dog blood was controlled. In
order to perform a direct comparison between methods, blood samples for all three measurements
(plasma-Hitachi 917, serum-Hitachi 917, whole blood-One Touch Ultra) were collected simultaneously
from the same site (jugular vein) in the same animals. Absolute values for glucose concentration
differed between methods of measurement and matrices. However, linear correlation was observed
between glucose levels measured in whole blood using One Touch Ultra and in plasma (R2=0.93,
Figure 1) or in serum (R2=0.93, Figure 2). In addition, the results of the study confirmed that although
glucose values obtained for plasma and for serum were similar, measurements in these two matrices
could not be used interchangeably. As there was a clear linear correlation (R2=0.96, Figure 3), conversion between plasma and serum glucose could be done using the corresponding equation.
Comment 1
LO
>2.1
Extremely low
1.1-1.9
2.1-3.0
Very low
1.9-2.3
3.0-3.5
Low
2.3-3.2
3.5-4.5
Somewhat low
3.2-5.9
4.5-7.5
Normal
1 – Suggested interpretation, may be interpreted differently dependent on the status of the animals.
Comparison of the results of blood sampling from different sites
In humans, One Touch Ultra is predominantly utilised for measurements of capillary blood. Preliminary tests had shown that it is practically impossible to get any notable amount of blood by
puncturing the dog skin. Therefore, sites at the leg and at the ear at which veins are coming close
to the skin surface were chosen as alternative sites of blood sampling (sparing the jugular vein from
multiple puncturing). In order to perform a direct comparison between the sites, blood samples were
collected simultaneously from all three sites from the ear (auricular vein), from the leg (cephalic vein)
and, from the neck (jugular vein, used as a reference). Measurements of blood collected from the
three sites gave very similar results (see Figure 4) and, based on the good quality of the correlation,
it is concluded that the sites can be used interchangeably within the same study. However, blood
sampling from the ear was regarded as less appropriate for multiple sampling, as it was more traumatic for the sampling area, and resulted in somewhat higher spreading of the results, particularly in
the normoglycemic animals. Therefore, it was concluded that the cephalic vein is a good alternative
for blood sampling for glucose measurements using One Touch Ultra.
Repeatability of the results
Multiple measurements showed good repeatability of results. According to the information from
manufacturer of One Touch Ultra, variation of 20% is regarded as acceptable. Triplicate measurements of the same blood sample showed good repeatability of the results (see Table 2 below). Based
on these data, repeatability was calculated to be 4.6%.
Table 2: Repeated measurements of glucose using One Touch Ultra
Animal
No
1st measurement,
mmol/L
2nd measurement,
mmol/L
3rd measurement,
mmol/L
Mean,
mmol/L
CV%
Male 1
3.5
3.4
3.4
3.4
1.7
Male 2
1.3
1.3
1.3
1.3
0
Female 1
1.7
1.7
1.9
1.8
6.5
Female 2
1.4
1.7
1.6
1.6
9.8
Figure 3
Figure 1
Glucose (mmol/L)
Whole blood (One Touch) vs. plasma (Hitachi)
Glucose (mmol/L)
Serum vs. plasma (Hitachi)
7
5
7
y = 0.91456 + 1.11549x
R2 = 0.9279
y = 0.42220 + 1.01354x
R2 = 0.9607
6
5
Plasma
6
Hitachi
Corresponding plasma glucose, mmol/L
4
3
4
3
2
2
1
1
1
2
3
4
5
6
1
2
3
One Touch
4
5
6
7
Serum
Figure 4
Figure 2
Glucose (mmol/L)
Whole blood (One Touch) vs. serum (Hitachi)
Glucose concentration measured in blood collected
from different sites
7
Glucose, mmol/L
Hitachi
5
y = 0.55159 + 1.07205x
R2 = 0.9349
6
5
4
3
2
4
Ear (auricular vein):
y = -0.05421 + 0.94979x
R2 = 0.9084
3
2
Leg (cephalic vein):
y = -0.01294 + 0.98365x
R2 = 0.9604
1
1
1
2
3
4
5
6
1
One Touch
2
3
4
5
Glucose, sample collected from jugularis vein, mmol/L
For the studies where One Touch Ultra measurements are to be employed, care has to be taken to use
the correct conversion equation (for serum or for plasma, study specific) when comparing with the
other data (e.g. measurements performed using Hitachi 917).
Limits of detection for One Touch Ultra in dogs
According to the manufacturer, One Touch Ultra can detect human blood glucose levels in the range
of 1.1-33.3 mmol/L. Upper limit of detection in dogs was outside the scope of the present study.
Using the corresponding equations (Figures 1 and 2), the lower limit of detection of One Touch Ultra
(1.1 mmol/L) was found to correspond to dog plasma glucose level of 2.1 mmol/L and to dog serum
Conclusion
One Touch Ultra glucometers can be used successfully for monitoring of glucose status in dogs,
resulting in less stress for the animals, smaller blood sampling volume, and faster delivery of
results.
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