Reference range of the IGF-1
concentration in feline plasma using
an immunochemiluminescence assay
Y. Raiman, 3382982
Tuturs: Dr. H.S. Kooistra, Dr. Ir. J.A. Mol, Drs. M. Prins
Veterinary Medicine, Utrecht University
17-12-2014
Abstract
In 20-25% of all cats with diabetes mellitus the diabetes is secondary to a different
disease, for example hypersomatotropism. This disease is caused by an anterior
pituitary tumour. For the optimal treatment, it is important to find the underlying cause
of the diabetes. The plasma insulin-like growth factor-1 (IGF-1) concentration is a
reliable indicator for hypersomatotropism. The current method, i.e. radioimmunoassay,
uses radioactive material, only takes place when enough samples are received and is
costly. With the new method, the immunochemiluminescence assay, the waiting period
will be significantly shorter, radioactive material will not be used and the costs would be
lower. The goal of this research is to find a reference range for the IGF-1 concentration
in feline plasma samples measured with an immunochemiluminescence assay.
92 plasma samples were selected from leftover feline plasma samples sent to the UVDL
between January and October 2014 for measurements not related to IGF-1. 42 samples
were sent in by veterinarians of the university clinic and 50 samples were sent in by
external first-opinion veterinarians in the Netherlands. The selection criteria were >6
years of age, with no kidney or liver disease, hypersomatotropism or diabetes in their
medical history. All the samples were measured using the Immulite 2000® (Siemens)
preceded by extraction with ethanol-formic acid to separate the IGF-1 molecules from
its ternary complex with IGFBP and ALS.
Within the 92 samples, 7 had a plasma IGF-1 concentration of >900ng/ml. Of these
samples, 5 were excluded from the study because the patients were previously
diagnosed with kidney disease. Using a box-and-whiskers plot with a 95% right-sided
reference interval, the reference range was calculated to be <795ng/ml.
Using this method and its reference range, it will be possible to measure the plasma IGF1 in patients’ blood samples on a weekly basis. Future research can be performed to
optimise the sample group by using only healthy individuals and reducing the amount of
variables between samples.
Introduction
Diabetes mellitus (DM) is a common
problem in cats. The majority of feline
patients with DM has a type similar to
diabetes
mellitus
type
2
in
Rijnberk2010
humans.
In approximately 2025% of cats the DM is secondary to a
different endocrinological condition, e.g.
hypersomatotropism
or
hypercortisolism, and is called type 3
diabetes mellitus. In these cats the
veterinarian and owners find it difficult
to regulate the diabetes and high
dosages are often necessary. For these
cats further diagnostic testing is
indicated to search for a primary
endocrinological disease. For future
management of the disease it is vital to
know whether an additional therapy for
the primary disease could aid in the
treatment of the patient.
The focus of this research will be on
hypersomatotropism as the underlying
disease causing secondary DM and more
importantly a new method of measuring
will be discussed and its reference range
will be determined.
In cats hypersomatotropism is caused
by a pituitary tumour Rosca2014 and is
mostly seen in castrated, male cats of 6
to 15 years old.Rijnberk2010 This condition
is characterised by hypersecretion of
growth hormone (GH) by the anterior
pituitary. GH excreted by the anterior
pituitary has a direct and an indirect
anabolic effect. Pierzchała2012 It promotes
hepatic synthesis of insulin-like growth
factor 1 (IGF-1).Gonzalez2014, Pierzchała2012
Excretion of IGF-1 from the body occurs
through the urinary pathway.Nam1990 IGF1 mediates many functions of GH, for
instance
cell
growth
and
proliferation.Sandhu2002 Symptoms in cats
with
hypersomatotropism
are
polyphagia, polyuria, polydipsia, weight
gain, lameness, enlargement of the paws
and facial features. Secondary to
hypersomatotropism diabetes mellitus
is generally present.
For
the
diagnosis
of
hypersomatotropism either the plasma
GH or the plasma IGF-1 is measured.
Measuring GH has proven to be
unreliable, as it is secreted in
pulses.Reusch2006
For
a
reliable
measurement of the GH concentration
three to five blood samples are needed.
The concentration of IGF-1 in the plasma
is less subject to fluctuations. Therefore
a single blood sample is sufficient for a
reliable measurement of the IGF-1
plasma concentration. Follow up testing,
after an outcome of IGF-1 plasma
concentration higher than the reference
range, is imaging of the pituitary using
an MRI or CT scan.Roasca2013
In cats, as in many other mammals, most
IGF-1 molecules in the plasma form a
ternary complex with IGFBP3 (insulinlike growth factor binding protein 3)
and ALS (acid-labile subunit).Lewitt2000
Most of the IGF-1, IGFBP3 and ALS are
synthesised in the liver.Wu2010 This
complex hinders IGF-1 from leaving the
circulation to the tissues and therefore
increases its halflife.Zapf1995,Lewitt2000 It is
proven that a direct assay, i.e. without
separating the complex made up of IGF1, IGFBP3 and ALS, results in falsely high
concentrations of IGF-1. Tschuor2012
Previous research has shown that
separation of this complex with ethanolformic
acid
extraction,
thereby
removing IGFBP3 and ALS from the
sample prior to analysis, provides the
most reliable measurements in feline
plasma.Baxter1989, Blum 1994
RIA (radioimmunoassay) has been
widely used for the measurement of IGF1. Tschuor2012,Berry2003,Castigliego2011
This
method involves radioactive substances
for which special measures need to be
taken in the laboratory. For this reason
RIA is not performed in the UVDL
(University
Veterinary
Diagnostic
Laboratory), but in a separate
laboratory (located at the veterinary
faculty in Utrecht, the Netherlands). In
this laboratory RIA will only be
performed when enough samples are
received, resulting in a long waiting
period for the results.
The new method, based on an
immunochemiluminescence assay using
the Immulite® 2000 (Siemens) preceded
by an extraction of IGF-1 from IGFBP
and the ALS using ethanol-formic acid,
will take place in UVDL on a weekly
basis, making the waiting time for a
result considerably shorter and the costs
lower.
The goal of this research is to find a
reference range for the insulin-like
growth factor-1 concentration in feline
plasma samples measured by the
Immulite® method preceded by an
extraction with ethanol-formic acid.
Material and method
Samples
In the period between January and
October 2014 leftover feline plasma
samples, containing more than 0.5ml of
plasma, of cats >6 years old and sent to
the university laboratory for any
measurement, were evaluated. 92
samples were selected. 42 samples were
sent in by veterinarians working at the
university clinic and 50 samples by
veterinarians from first opinion clinics
in the Netherlands. The plasma samples
were sent in for measurements not
related to hypersomatotropism and
diabetes mellitus. At the university
laboratory these samples were stored at
-20°C. Using the medical history of the
university patients, a selection of
suitable plasma samples was made.
Patients known with diabetes mellitus,
hypersomatotropism and liver and
kidney problems were excluded. There
was no medical history available of the
external patients. From these samples a
selection was made using the available
information. After measurement of IGF1 external veterinarians were contacted
for information about the medical
history of their patients when a high
plasma IGF-1 concentration (>900ng/ml
IGF-1) was found. The generally used
upper limit for IGF-1 is 1000ng/ml.
Tschuor2012
Extraction and measuring method
Extraction fluid: 87.5% EtOH/12.5%
formic acid. Add to 11.6ml MilliQ, 938µl
formic acid and add absolute EtOH to
make the volume of the solution 100ml.
Combine 100µl plasma with 400µl
extraction fluid. Cover the tubes with
parafilm and foil, vortex and leave for 30
minutes
at
room
temperature.
Centrifuge 30 minutes at 5000rpm and
4°C (pipet as soon as possible). This
equals to a G-force of 5000g.
For the measurements of the samples an
Immulite® 2000 (Siemens) was used.
This is a solid-phase, enzyme-labeled
chemiluminescent immunometric assay.
Only serum and heparin samples should
be used in this assay, EDTA would affect
the results. The required sample volume
is 20µl. The IGF-1 bead pack contains
200 beads coated with murine
monoclonal anti-IGF-1. The IGF-1
reagent wedge contains 6.5ml alkaline
phosphate (bovine calf intestine)
conjugated to polyclonal rabbit anti-IGF1 in buffer and 6.5ml IGF-2 in
buffer.Instructions Immulite®
Statistics
The coefficient of skewness, the
coefficient of kurtosis and the
D'Agostino-Pearson test were used to
determine whether the data had a
normal distribution.
A box-and-whisker plot was used to
calculate
the
reference
range.
Additionally the confidence interval of
the reference range was calculated.
3
Results
Of the 7 samples from external
veterinarians with an IGF-1 plasma
concentration of >900ng/ml, 5 were
said to have a kidney problem in their
medical history and for this reason these
samples were excluded from this
research.
0
200
400
Statistically the data set does not have a
normal distribution. The coefficient of
skewness, the coefficient of kurtosis and
the D'Agostino-Pearson test for normal
distribution reject a normal distribution.
Using a box-and-whiskers plot (see
figure 1) with a right-sided reference
interval of 95%, the upper limit of the
reference range of the IGF-1 plasma
concentration is calculated to be
795ng/ml. This limit lies within a 90%
confidence interval from 645 to
1090ng/ml (see figure 2).
600
800
IGF-I (ng/ml)
1000
1200
Figure 1. Box-and-Whiskers plot
4
1200
1000
800
600
Methods
Percentile
400
200
0
Figure 2. Confidence interval
Discussion
The plasma samples used in this study
are all leftover from plasma sent in by
veterinarian for an array of reasons. One
can assume that most samples were sent
in because the cats were showing
symptoms of some kind. For this reason
these samples might not be an ideal
representation of the healthy Dutch cat
population.
Ideally
only
healthy
individuals are used for determining a
reference range.
and were therefore excluded from this
study. We assume the other patients,
that were included and that had a
plasma IGF-1 concentration of less than
900ng/ml, were good representatives.
Further research using only samples
from the university clinics might be
more reliable as one could see the full
medical history. Ideally using healthy
members of the cat population would be
most suitable to determine a reference
range.
The medical history of most of the
external patients was unknown. Of these
patients only the ones with an unusually
high IGF-1 plasma concentration were
investigated further by contacting the
veterinarian who sent the sample to the
university laboratory. 7 samples from
external patients were higher than
900ng/ml, 5 of these patients have
kidney disease in their medical history
The time between collecting the
samples, centrifuging them and cooling
them is unknown for every sample as
each sample was sent in by either
university veterinarians or first opinion
veterinarians
without
a
specific
protocol. The time between collecting
the blood and arriving at the university
laboratory is also unknown. As is the
temperature in which the samples were
5
stored and sent. The vein from which
the samples were taken is also
unknown. These variable might have an
effect on the IGF-1 concentrations in the
plasma samples. Further research might
shed light on the matter.
When determining a reference range
individuals of a similar age group to that
specific disease should be used, in this
case patients with hypersomatotropism
are adult cats. For this reason samples of
young cats were excluded from this
study. It is said that young animals have
a higher plasma IGF-1 concentration
than adult animals.Juul1995
More research can be done to further
simplify the extraction method to make
the process even more user friendly.
Preliminary research has shown that
shortening the centrifuge time from 30
minutes down to 5 minutes and
changing the temperature during
centrifuging from 4°C to 20°C results in
similar measurements of the IGF-1
plasma concentration.
Kidney problems seem to have a strong
effect on plasma IGF-1 concentration in
cats. Of the 7 cats with a plasma IGF-1
concentration of >900ng/ml, 5 were
known to have a kidney problem. More
research can be done to explore the
relation between kidney problems and
IGF-1 plasma concentrations.
A normal distribution of the data set was
rejected using several tests. The reason
for the absence of normal distribution
can be explained by examining the boxand-whiskers plot. 23 of the total of 92
samples
had
a
plasma
IGF-1
concentration of <25ng/ml. Within this
study the reason for this low outcome is
unknown. Further research might
explain the reason for this skewed
distribution.
Conclusion
With the plasma samples used in this
study a reference range was calculated
within a confidence interval. The
reference range is <795ng/ml within the
90% confidence interval
6451090ng/ml. There are several issues
that need to be addressed in future
research. For following studies the
advice is to use a group of healthy cats
that form a good representation of the
disease population, for example keeping
age as a criterion for inclusion. A
reference range, made using these
individuals, would be more reliable for
comparison to patients with diabetes
mellitus that might have primary
hypersomatotropism caused by a
tumour in the anterior pituitary. Using a
protocol for collection of the blood
samples and centrifuging and storing of
the plasma samples will decrease the
amount of variables between the
samples. This study is the first step in
providing an insight in the reference
range for plasma IGF-1 using an
immunochemiluminescence assay. In
the future this method could replace the
currently used method i.e. RIA.
6
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