Confused about Lipase Assays?
- During pancreatitis, pancreatic lipase is released into the
bloodstream and can be used as a diagnostic marker for the
- Beyond the pancreas, lipases are released from various other
organs, adding to the total serum lipase activity.
- Spec cPL® and Spec fPL® specifically measure lipases of
pancreatic origin, making them very sensitive and specific for a
diagnosis of pancreatitis.
- DGGR- and triolein based lipase assays measure the total serum
lipase activity and thus are unspecific.
- Spec cPL® and Spec fPL® remain superior for the diagnosis of
pancreatitis in both dogs and cats.
Acute or chronic pancreatitis are now recognized to be common in
both dogs and cats, but their diagnosis can be challenging. Various
diagnostic tests have been used to confirm a diagnosis of acute and
chronic pancreatitis, including the measurement of serum lipase
activity, measurement of serum pancreatic lipase immunoreactivity
(PLI), abdominal ultrasound, and histopathology.
Assays for the measurement of serum lipase activity were
commonly used in the past because they were minimally invasive,
inexpensive, and did not require special equipment or expertise.
Although the exocrine pancreas is a major source of the serum
lipase activity, it is not the only organ or type of cell that
produces, stores, and releases a lipase. Lipase may be released by
the stomach (gastric lipase), the liver (hepatic lipase),
endothelium (endothelial lipase), and by many other organs and
cells. All of these lipases contribute to the total lipase activity
measured in serum. Thus, assays for the measurement of serum total
lipase activity lack specificity for the pancreas. In addition,
they are not specifically sensitive for the diagnosis of
pancreatitis. Depending on the cutoff value used, total serum
lipase activity has been reported to have a sensitivity as low as
13.6% for macroscopic pancreatitis, thus missing up to 86.4% of
patients with pancreatitis.1 The sensitivity and
specificity naturally changes with different cut-off values. This
explains why some studies found higher sensitivities (up to 71%)
but at the expense of specificity with an increasing number of
false-positive results (specificity 43%, i.e. 57% of positive
results are false-positive).2 Furthermore,
renal,3,4 gastrointestinal,5 hepatic, and
neoplastic disease,6 as well as steroid
administration7 have all been shown to cause increases
in total serum lipase activity.
Serum pancreatic-lipase immunoreactivity (PLI) as measured by
the Spec cPL® or Spec fPL® specifically measure lipase that
originates from the acinar cells of the canine or feline pancreas,
respectively. These assays use antibodies directed against
pancreatic lipase and to date no other lipase has been shown to
cross-react with these antibodies.8-10 Therefore, Spec
cPL® and fPL® are considered to be the most specific diagnostic
tests for the exocrine pancreas. Also, measurement of Spec PL® is
highly sensitive for a diagnosis of pancreatitis in both dogs and
cats. In dogs with clinically significant signs of pancreatitis,
Spec cPL® has been shown to identify the disease with a sensitivity
of 82 to 94%. In dogs with less severe pancreatitis, Spec cPL®
still showed the highest accuracy among any diagnostic test with a
sensitivity of 64%.1 In cats, Spec fPL® correctly
identified patients with pancreatitis with a sensitivity between
54% (subclinical to mild disease) to 100% (moderate to severe
disease). Specificities for Spec cPL® and fPL® are even higher and
have been reported to be between 79 and 100%. Thus, these tests
rarely produce false-positive results. This is especially important
as many other diseases may cause clinical signs similar to those
seen with pancreatitis.
Recently, two substrates for the measurement of total serum
lipase activity (triolein and 1,2-o-dilauryl-rac-glycero-3-glutaric
acid (-6’-methylresorufin, DGGR)) have been marketed for the
measurement of total serum lipase activity. Both substrates are
used in catalytic assays, where serum lipases cleave the substrate
and products of this reaction are measured via colorimetry. The
v-LIP-P slide detects serum lipases using triolein as the reaction
substrate and a negatively charged detergent as an auxiliary
agent11 while the DGGR-lipase assays use DGGR as a
substrate.12 Both substrates have been claimed to have a
comparable diagnostic utility to that of serum Spec PL.
In 2005, Graca et al. validated a DGGR lipase assay for use in
dogs.13 Their study showed, that using a cut-off value
of 120 U/L, this test had a high sensitivity of 93%, but poor
specificity of 53%. When the cut-off value was increased to 180
U/L, similar to the currently used “gray zone” approach to
interpret Spec cPL® results, the specificity slightly improved
reaching 66%, whereas sensitivity decreased to 73%. The authors of
that study discussed the possibility of cross-reactivity of
non-pancreatic lipases with the substrate. Indeed, a recent study
by our group indicates that DGGR is hydrolyzed by other enzymes
(likely non-pancreatic lipases) (unpublished data). Serum lipase
activity in leftover serum samples of 48 dogs with exocrine
pancreatic insufficiency (TLI ≤ 1 mg/L) and 66 healthy control dogs
was measured using a DGGR-based assays (Diazyme Laboratories,
Poway, CA and Stanbio Laboratory Boerne, TX). Our data showed that
serum lipase activity was within the reference interval in 33 of 48
dogs with EPI using a DGGR-based assay (Figure 1).
Observation in Figure 1:
Serum lipase activity measured by a DGGR-based assay was readily
detectable in many dogs with EPI.
Conclusion - DGGR-based assays for the measurement of serum
lipase activity are not specific for pancreatic lipase.
Figure 1. Serum lipase activities in dogs with EPI and
healthy control dogs as measured by an assay utilizing DGGR as a
The center lines show the medians for each group and the
whiskers display the interquartile ranges
Similarly, we compared serum lipase activity measurements in
leftover serum samples from 50 dogs with EPI (TLI ≤ 2.5 mg/L) and
50 healthy control dogs using the v-LIP-P slide (FujiFilm
Corporation, Tokyo, Japan) and the Spec cPL® (IDEXX Laboratories,
Inc., Westbrook, Maine, USA). While the serum Spec cPL® was in the
lower 20% of the reference interval in 49 of 50 (98%) dogs with
EPI, serum lipase activity measured with the v-LIP-P slide was in
the lower 20% of the reference interval in only 29 of 58 EPI dogs
(58%), indicating the detection of lipases other than that of
pancreatic origin (Figures 2 and 3). These results clearly show
that, in contrast to Spec cPL®, triolein and DGGR are not specific
for the measurement of pancreatic lipase.
Observation in Figure 2 and 3:
Spec PL immunoassay concentrations are very low or undetectable
in serum from dogs with EPI, but lipase activities are readily
detectable and often normal in the same samples.
Conclusion – Spec PL is pancreas specific in origin, but lipase
activity as measured by the v-LIP-P slide is not.
Figure 2: Lipase activity as measured by thev-LIP-P
slide in 50 healthy dogs and 50 dogs with EPI.
Many of the dogs with EPI had significant serum lipase
activities. The lines indicate the medians for both groups.
Figure 3: Pancreatic lipase immunoreactivity as measured
by Spec cPL in 50 healthy dogs and 50 dogs with EPI.
There is much better separation between healthy dogs and dogs
with EPI than for the v-LIP-P slide. The lines indicate the medians
for both groups.
Various studies have been reported that directly compared the
aforementioned assays with the Spec cPL® or
fPL®.11,14,15 All of these studies found good to
moderate correlation between the assays. However, while it is
intuitive to assume that serum lipase activity increases as the
fraction of pancreatic lipase increases in patients with
pancreatitis and that all aforementioned assays will detect that
increase in activation, a correlation between tests is not
equivalent to a test having the same sensitivity and specificity
and thus discriminatory power between disease and controls. A
recent study found a moderate agreement (Cohen´s k value 0.803)
between a DGGR lipase assay and Spec cPL® when a 2-fold DGGR lipase
“gray zone” was applied with cut offs of 216 U/L for DGGR and 400
mg/L for Spec cPL®. Sensitivity and specificity could not be
calculated since pancreatic histopathology was not
available.14 Another study compared serum lipase
activity as measured with the v-LIP-P slide (FUJI DRI-CHEM 7000V,
FUJIF- ILM corporation, Tokyo, Japan) with the measurement of
pancreatic lipase using the Spec cPL® and showed good correlation
(r=0.91). However, when looking at the data, the graph clearly
showed good correlation for lower lipase values, but increasing
variation with increasing values, starting at about 400 mg/L Spec
cPL® (cutoff for pancreatitis) (see Ishioka, K., Hayakawa, N.,
Nakamura, K. & Terashima, K. Patient-side assay of lipase
activity correlating with pancreatic lipase immunoreactivity in the
dog. J. Vet. Med. Sci. 73, 1481–1483 (2011)
. This indicates non-constant variances (heteroscedasticity) and
thus a simple correlation does not reflect the assays’ true
A recent study comparing the DGGR lipase assay (cutoff > 26
U/L) with Spec fPL® (> 3.4 mg/L) in cats with acute or chronic
pancreatitis also showed poor agreement between the two assays
(Cohen´s k value 0.601)15. Based on histopathology,
sensitivity and specificity were 48% and 63% for the DGGR lipase
assay and 57% and 63% for Spec fPL®, respectively. However, a
previous study showed higher sensitivities and specificities for
Spec fPL® in cats with different stages of
pancreatitis.16 In cats, with histopathologically
confirmed mild pancreatitis, sensitivity of Spec fPL® was 54%,
while this percentage increased to 100% in moderate to severe cases
of pancreatitis. Specificity of Spec fPL® has been as high as 100%
when tested against a healthy control group. More importantly,
specificity of Spec fPL® reached 67% in cats with clinical signs
mimicking pancreatitis but with normal pancreatic
Effect of influence factors on results of lipase
- The triolein-based lipase assay v-LIP-P slide is highly
influenced by hemolysis, lipemia, and icterus
- In contrast, hemolysis, lipemia, and icterus have no effect on
results of the Spec cPL® assay
In addition to significant problems with sensitivity and
specificity, assays for total serum lipase activity have been shown
to be greatly influenced by hemolysis, lipemia, and icterus,
influence factors that are frequently observed in dogs with
pancreatitis and/or other gastrointestinal or hepatic diseases. In
an in-vitro study, we recently evaluated the effects of these
factors on the performance of lipase activity assays using the
substrate triolein, and on the performance of the Spec cPL® ELISA
assay. Serum samples that were spiked with canine hemoglobin,
Intralipid®, and synthetic ditaurobilirubin, mimicking hemolysis,
lipemia, and icterus, respectively (unpublished data). Evaluation
of the v-LIP-P slide showed that this assay significantly
underestimates serum lipase activity in hemolyzed and icteric
samples, thus limiting sensitivity of the assay in these samples.
This is likely to translate into a high false-negative rate in
patients with pancreatitis, i.e., missing a diagnosis of
pancreatitis in many patients. In lipemic samples the use of
triolein dramatically increased the reported lipase activity,
potentially leading to a dramatically increased rate of
false-positive diagnosis of pancreatitis (poor specificity). In
contrast, hemolysis, lipemia, and icterus at the same levels showed
no effect on the performance of the Spec cPL® assay. These results
show, that the Spec cPL® and fPL® are superior for the diagnosis of
pancreatitis in dogs and cats to any lipase activity assay,
regardless of the substrate that is being used.
- Steiner JM, Newman S, Xenoulis P, Woosley K, Suchodolski J,
Williams D, et al. Sensitivity of serum markers for pancreatitis in
dogs with macroscopic evidence of pancreatitis. Vet Ther.
- Trivedi S, Marks SL, Kass PH, Luff JA, Keller SM, Johnson EG,
et al. Sensitivity and specificity of canine pancreas-specific
lipase (cPL) and other markers for pancreatitis in 70 dogs with and
without histopathologic evidence of pancreatitis. J Vet Intern Med.
2011 Jan 1;25(6):1241–7.
- Strombeck DR, Farver T, Kaneko JJ. Serum amylase and lipase
activities in the diagnosis of pancreatitis in dogs. Am J Vet Res.
- Polzin DJ, Osborne CA, Stevens JB, Hayden DW. Serum amylase and
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- Archer FJ, Kerr ME, Houston DM. Evaluation of three pancreas
specific protein assays, TLI(trypsin-like immunoreactivity), PASP
(pancreas specific protein) and CA 19-9 (glycoprotein) for use in
the diagnosis of canine pancreatitis. Zentralbl Veterinarmed A.
- Quigley KA, Jackson ML, Haines DM. Hyperlipasemia in 6 dogs
with pancreatic or hepatic neoplasia: evidence for tumor lipase
production. Vet Clin Pathol. 2001;30(3):114–20.
- Parent J. Effects of dexamethasone on pancreatic tissue and on
serum amylase and lipase activities in dogs. J Am Vet Med Assoc.
- Steiner JM, Berridge BR, Wojcieszyn J, Williams DA. Cellular
immunolocalization of gastric and pancreatic lipase in various
tissues obtained from dogs. Am J Vet Res. 2002
- Steiner JM, Rutz GM, Williams DA. Serum lipase activities and
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- Neilson-Carley SC, Robertson JE, Newman SJ, Kutchmarick D,
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- Ishioka K, Hayakawa N, Nakamura K, Terashima K. Patient-side
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immunoreactivity in the dog. J Vet Med Sci. 2011
- Panteghini M, Bonora R, Pagani F. Measurement of pancreatic
lipase activity in serum by a kinetic colorimetric assay using a
new chromogenic substrate. Ann Clin Biochem. 2001 Jul;38(Pt
- Graca R, Messick J, McCullough S. Validation and diagnostic
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for the diagnosis of acute pancreatitis in dogs. Vet Clin Path.
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