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SNAP Tests for Pancreatitis in Dogs and Cats: SNAP Canine Pancreatic Lipase and SNAP Feline Pancreatic Lipase
Author’s Accepted Manuscript SNAP Tests for Pancreatitis in Dogs and Cats: SNAP cPL and SNAP fPLSNAP tests for pancreatitis Panagiotis G. Xenoulis, Jörg M. Steiner www.elsevier.com/locate/yspsu
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To appear in: Topics in Companion Animal Medicine Cite this article as: Panagiotis G. Xenoulis and Jörg M. Steiner, SNAP Tests for Pancreatitis in Dogs and Cats: SNAP cPL and SNAP fPLSNAP tests for p a n c r e a t i t i s , Topics in Companion Animal Medicine, http://dx.doi.org/10.1053/j.tcam.2016.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
SNAP tests for pancreatitis in dogs and cats: SNAP cPL and SNAP fPL
Panagiotis G. Xenoulis1,2, Jörg M. Steiner3
1
Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece
2
Animal Medical Center of Athens, Athens, Greece
3
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of
Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA
Short title: SNAP tests for pancreatitis
Key words: pancreatic lipase; pancreatitis; PLI; Spec cPL; Spec fPL
Correspondence: Dr. Panagiotis Xenoulis, Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, 224 Trikalon, Karditsa, 43100, Greece Email: [email protected], Tel. +30 24410 66085.
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Abstract A clinical diagnosis of pancreatitis in dogs and cats can be challenging. Several diagnostic modalities have been evaluated over the years for the diagnosis of canine and feline pancreatitis, but the majority of these modalities have been shown to be of limited clinical usefulness because of poor performance, limited availability, and/or because they are invasive. Assays for the measurement of pancreatic lipase immunoreactivity (Spec cPL in dogs and Spec fPL in cats) were first developed about 15 years ago, and studies have shown that they are currently the serum tests of choice for the evaluation of canine and feline patients, respectively, suspected of having pancreatitis. This is a direct consequence of their high specificity of detecting only pancreatic lipase and their sensitivity for pancreatitis when compared to other serum tests. SNAP cPL and SNAP fPL are in-clinic tests that have been developed based on the Spec cPL and Spec fPL assays. As with any other test, false positive and false negative results do occur with pancreatic lipase immunoreactivity assays and it is important to know the limitations of these assays.
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Introduction Accurate clinical diagnosis of pancreatitis in dogs and cats can be a big challenge in small animal practice. Several blood tests have been developed and evaluated for the diagnosis of canine and feline pancreatitis over the past decades, but most of them have been proven to be inadequate.1 In addition, several imaging modalities have been evaluated for the diagnosis of canine and feline pancreatitis, and with the exception of ultrasonography, none has been shown to be both valuable and practical for routine use for that purpose.1 During the last 15 years, pancreatic lipase immunoreactivity (PLI) assays and their companion SNAP PL (SNAP pancreatic lipase) tests have been developed, validated, and evaluated for the diagnosis of pancreatitis in dogs and cats, and have been proven to be useful.1,2 The present review will focus on SNAP cPL (SNAP canine PL) and SNAP fPL (SNAP feline PL) tests. A detailed review on the development and clinical use of canine and feline PLI assays has recently been published.2
Historical background: the need for development of new assays for canine and feline pancreatitis Assays for the measurement of serum amylase and lipase activities were initially developed more than 100 years ago, and were the first serum markers of pancreatic disease in humans (initially) and several animal species (much later).1,3,4 The application of these assays in small animal clinical practice did not start gaining popularity until the early 1960s, but at that time, information on the performance of those assays for the diagnosis of pancreatitis in dogs and cats was very limited.3 It was soon evident that the use of serum amylase and lipase activities as tests for the diagnosis of pancreatitis was associated with several inherent limitations.1 Several studies, both experimental and clinical, in dogs and cats showed that serum lipase and amylase 3
activities lack both sensitivity and specificity for the diagnosis of pancreatitis.1,5-12 Subsequently, it was shown that many tissues other than the pancreas (e.g., gastric mucosa, hepatic parenchyma, and many others) synthesize and release amylases and lipases.1,9,13-15 Also, generally speaking, catalytic assays cannot differentiate amylases and lipases according to their tissue of origin, which requires broadening the reference interval of these assays and thus not only accounts for the limited specificity (around 50% for both), but also the limited sensitivity for the measurement of serum amylase and lipase activities for pancreatitis in dogs.1,5-10,13,16 The reported sensitivity of serum amylase and lipase activities for spontaneous canine pancreatitis varies but is generally low (32% - 73% for lipase activity and 41% - 69% for amylase activity).1,10,17,18 In cats, both serum lipase and amylase activities do not appear to be of any clinical value in the diagnosis of spontaneous feline pancreatitis.1,12,19,20 Trypsin-like immunoreactivity (TLI) assays measure trypsinogen and trypsin in serum and were initially developed as more sensitive and specific markers of pancreatic disease in dogs and cats.21-24
30,31 33,34
These markers have become the gold standard for the diagnosis of
exocrine pancreatic insufficiency (EPI) in dogs22 and cats25 but studies have shown their sensitivity for the diagnosis of pancreatitis to be low for both dogs (36%-47%)10,26 and cats (28% - 64%).16,27,28 Several other diagnostic markers for pancreatitis have been developed and studied (e.g., serum concentrations of pancreatic elastase-1,29 phospholipase A2,30 trypsin-α1-antitrypsin complexes,31,32 and α2-macroglobulin,33,34 or plasma and urine concentrations of trypsinogen activation peptide (TAP)16,26), but none of these can currently be recommended for routine use in dogs and cats, either because their diagnostic performance has not been sufficiently evaluated clinically, or because they are associated with a limited sensitivity and/or specificity. Also, most of these assays are not routinely available.
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Pancreatic lipase As mentioned above, many tissues other than the pancreas (e.g., gastric mucosa, hepatic parenchyma, and others) synthesize and release lipases, which all share the same function (i.e., hydrolysis of triglycerides).1,9,13-15 This, in association with the limited ability (depending on the assay characteristics and conditions) of most traditional catalytic assays to differentiate lipases according to their tissue of origin results in low specificity and sensitivity of most serum lipase activity assays for pancreatitis in dogs and cats.1,5-14,17-20 Pancreatic lipase, which is of interest for the evaluation of the exocrine pancreas, is believed to be exclusively expressed by pancreatic acinar cells. This is supported by the findings of an immunolocalization study in dogs, that showed that canine pancreatic lipase was only expressed by pancreatic acinar cells among 38 different types of tissues.35 Another study in dogs with EPI showed a nearly total absence of serum cPLI concentration, further confirming that pancreatic lipase is exclusively of pancreatic origin.11 Similar studies have not been yet reported in cats. Although all lipases, including pancreatic lipase, share the same function, lipases of different cellular origin are encoded by different genes and have different amino acid sequences from one another and thus generate a distinct immunological response.14,15 Therefore, pancreatic lipase may function similarly to, but it is structurally different from other lipases. This means that assays that detect specific protein molecules based on their unique tertiary structure, such as immunoassays, should be able to specifically measure pancreatic lipase with no interference from other lipases. In addition, in contrast to catalytic lipase assays, immunoassays for pancreatic lipase would be species-specific because the pancreatic lipase amino acid sequence differs among species.
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PLI and Spec PL assays: the basis of SNAP PL tests SNAP PL tests are based on the technology, properties, and characteristics of the Spec PL assays, which were developed based on the original PLI assays. Therefore, in order to get a better understanding of the SNAP PL tests it is necessary to provide some basic information on the Spec PL assays. Pancreatic-lipase immunoreactivity assays are immunoassays developed to specifically quantify lipase of pancreatic origin based on its unique tertiary structure, while not detecting lipases of other tissue origin.11,14,15,35,36 Therefore, the PLI assays have inherent advantages over the traditional serum lipase activity assays that make them more suitable for specific evaluation of the exocrine pancreas in dogs and cats. For the development of the PLI assays, canine and feline pancreatic lipases were purified from canine and feline pancreas, respectively, following a sequence of purification experiments.37,38 Canine pancreatic lipase was found to be a low molecular weight protein of approximately 50.7 kDa37, while feline pancreatic lipase was found to have a molecular weight of 52.4 kDa using mass spectrometry.38 Following purification, antiserum directed against canine or feline pancreatic lipases was developed in rabbits by repeated inoculation of purified canine or feline pancreatic lipase, respectively.39-41 Purification of polyclonal antibodies was accomplished by affinity chromatography, and purified antibodies were used for the development of a radioimmunoassay (RIA)39 and a “sandwich” ELISA40 for canine PLI (cPLI) and an RIA for feline PLI (fPLI).41 All 3 assays were analytically validated, and were found to be sensitive, linear, accurate, precise, and reproducible for clinical application.39-41
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The originally developed cPLI and fPLI assays were in-house immunoassays that used small-scale manufacturing processes and therefore had a limited availability. In order for those assays to become suitable for commercial application and broad use, the originally developed assays had to be modified. The original cPLI ELISA and fPLI RIA assays were used as gold standards for the development of the new canine and feline assays, respectively. The new assays were named Spec cPL® (Specific Canine Pancreatic Lipase) and Spec fPL® (Specific Feline Pancreatic Lipase), and released for wide clinical use in 2007 and 2008, respectively, at which time they replaced the original assays. The main differences between the new and the old assays were as follows:39-42 a) the original PLI assays used purified pancreatic lipase whereas the Spec PL assays use a recombinant peptide as an antigen b) instead of using polyclonal antibodies produced in rabbits, the new assays use monoclonal antibodies secreted by cell lines that were produced in Balb/C mice; c) the original cPLI assay used a horseradish peroxidase (HRP) detection system that was added to the ELISA plate as a separate step, while the Spec PL assays use a pre-labeled anti-pancreatic lipase HRP conjugate allowing for direct detection. In a recent validation study, the Spec cPL assay was reported to be precise and reproducible for clinical application.42 The comparison of the two canine assays (the original cPLI and the Spec cPL) showed general agreement but also illustrated that a positive bias existed, with approximately 50% higher concentrations for Spec cPL compared to the original cPLI assay.42 This bias was accommodated by adjustments of decision limits, which were also evaluated and confirmed clinically.18,42 The reference interval for the Spec cPL assay is 0-200 µg/L, with concentrations above 400 µg/L being considered consistent with pancreatitis. Concentrations between 200 µg/L and 400 µg/L are considered in the “gray zone”.
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In contrast to the Spec cPL assay, details on the development and validation of the Spec fPL assay have not yet been published in the peer reviewed literature. Based on the manufacturer, the assay is precise and reproducible for clinical application, and has a good agreement with the original fPLI RIA. The reference interval for the Spec fPL assay is 0-3.5 µg/L and concentrations above 5.3 µg/L are considered consistent with pancreatitis. As in the dog, a “gray zone” exists for Spec fPL and corresponds to concentrations between 3.5 µg/L and 5.3 µg/L.
Development and validation of SNAP cPL® and SNAP fPL® assays In clinical practice, the time required to obtain results using the Spec cPL and Spec fPL is at least 24 hours, and might be considerably longer if weekends or holidays are included. The same is true for countries where the Spec cPL and Spec fPL are not available. This turnaround time, although generally acceptable in many situations, is unsuitable for the diagnosis of acute and especially severe acute pancreatitis that would potentially benefit from an early diagnosis and management. Therefore, rapid, in-clinic, semi-quantitative, visually-read tests (SNAP tests) for the estimation of canine and feline pancreatic lipase immunoreactivity in serum were developed and are based on the same ELISA technology and monoclonal antibodies as the quantitative reference methods (Spec PL).43 The SNAP tests incorporate a reference spot that corresponds to the upper limit of the reference interval for each species (200 µg/L for the dog and 3.5 µg/L for the cat), and the color intensity of the sample spot is visually compared to the reference spot (Figure 1).43 Therefore, the results of this rapid assay are considered either normal (less intense than the reference spot) or abnormal (equally to or more intense than the reference spot). In the latter case, the actual
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pancreatic lipase immunoreactivity concentration may be in the “gray zone” (200 µg/L to 400 µg/L for Spec cPL or 3.6 µg/L to 5.3 µg/L for Spec fPL) or consistent with a diagnosis of pancreatitis (>400 µg/L for Spec cPL or >5.3 µg/L for Spec fPL) (Figure 2).43 This differentiation cannot be determined with certainty, however, using the in-clinic assay and further testing using the quantitative reference method is recommended. A recent validation study showed agreement of close to 100% between the SNAP cPL and the reference Spec cPL method for samples in the reference interval, with a slightly lower agreement (about 90%) for samples above the upper limit of the reference interval.43 Samples that are close to the upper limit of the reference interval (200 µg/L) are more likely to be incorrectly classified.43 The SNAP cPL assay was found to be precise and reproducible and therefore suitable for clinical use.43 Although studies on the validation of the SNAP fPL have not been reported in the literature yet, the manufacturer indicates that in an in-house study that included 80 cats this test had an agreement of 89 - 93% for samples with results within the reference interval and a 92 - 100% agreement for samples with results with Spec fPL concentrations above the upper limit of the reference interval. Therefore, similarly to the SNAP cPL test, this test appears to be suitable for routine clinical use.
Clinical performance characteristics of SNAP cPL The main use of SNAP cPL is to rule-out pancreatitis rather than diagnose it. This recommendation has its basis on the high sensitivity of the SNAP cPL test (between 80% and 95% for clinical acute pancreatitis), which means that a normal result makes diagnosis of pancreatitis very unlikely. Therefore, sensitivity of this test is more important than its specificity and the following discussion will focus mainly on the sensitivity of this test and its negative
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predictive value. Abnormal SNAP cPL results can be seen in dogs with Spec cPL concentrations in the gray zone or those with cPLI concentrations consistent with pancreatitis and further testing using the quantitative Spec cPL assay would be necessary in such cases. Therefore, diagnosis of pancreatitis cannot be based on the result of the SNAP cPL alone. Two clinical studies have been published in the peer reviewed literature that have both evaluated and compared the diagnostic performance of Spec cPL and SNAP cPL. The first of those studies included 84 dogs, 57 of which had a clinical presentation indicative of acute pancreatitis.18 In the absence of a diagnostic test with perfect accuracy, traditional referencebased methods for evaluating test sensitivity and specificity were considered by the authors to yield biased estimates. Therefore, latent class analysis was used to determine unbiased estimates of diagnostic sensitivity and specificity for the tests evaluated. Dogs were categorized in groups based on the probability of having pancreatitis after a panel of 4 internists reviewed each dog’s complete medical record, laboratory findings, and abdominal ultrasound findings. SNAP cPL was found to have a sensitivity that ranged between 91% and 94%.18 In the same study, the sensitivity of the Spec cPL assay (cutoff value of 200 μg/L) was very similar (86 - 94%).18 The conclusion of that study was that SNAP cPL and Spec cPL have a higher sensitivity for diagnosing clinical acute pancreatitis than does measurement of serum amylase or lipase activity and that SNAP cPL and Spec cPL have a good negative predictive value when there is low prevalence of disease. The second study included 38 dogs that presented with acute abdominal disease, 11 of which were diagnosed as having acute pancreatitis based on history, physical examination findings, and the results of complete blood count, biochemical analysis, and abdominal ultrasonography performed by a veterinary radiologist.44 The SNAP cPL test was found to have a
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sensitivity of 82%, while the sensitivity of Spec cPL was 70%. As in the previous study, there was a good overall agreement between SNAP cPL and Spec cPL (≥ 200 μg/L) for the entire cohort (κ = 0.78).44 In the same study, it was found that the SNAP cPL test can give up to 40% false positive results for pancreatitis, which underlines the recommendation that a diagnosis of pancreatitis should not be based on the results of the SNAP cPL alone.44 A third study that has only been reported in abstract form to date also evaluated SNAP cPL and Spec cPL for the diagnosis of pancreatitis.45 This study included 114 dogs with a clinical suspicion of pancreatitis. In this study, SNAP cPL and Spec cPL showed an agreement of 88% for dogs suspected to have pancreatitis. However, sensitivity and specificity of the SNAP cPL test were not specifically reported in that abstract. With the exception of the 3 above-mentioned studies, no other studies have directly evaluated the sensitivity of SNAP cPL for pancreatitis. However, several other studies have evaluated Spec cPL (and/or the original cPLI) for the diagnosis of pancreatitis as these assays have been available for much longer compared to the SNAP cPL test. As mentioned above, several studies have established that the SNAP cPL test has a high level of agreement with the Spec cPL assay,18,43 and therefore, the clinical performance characteristics of the Spec cPL and cPLI assays can provide a relatively accurate estimation of the performance characteristics of the SNAP cPL test. A series of studies10,46,47 on the sensitivity of cPLI for canine pancreatitis based on histopathology as a gold standard have been published. However, it is more challenging to accurately interpret and determine the clinical significance of the results of these studies, because the diagnosis of pancreatitis was primarily based on histopathological criteria. Therefore, clinically healthy dogs with histopathological lesions of the pancreas but clinically insignificant
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disease were also included in those studies.10,47 It is of note that in the most recent of those studies,47 in which 70 dogs euthanized for a variety of reasons were examined, 63 dogs (90%) had histopathologic evidence of pancreatitis of which 56 (89%) had only mild disease. In addition, two of those studies enrolled primarily47 or exclusively46 dogs with chronic pancreatitis. In the above mentioned 3 necropsy studies,10,46,47 the sensitivity of Spec cPL/cPLI ranged from 21% for mild (potentially clinically insignificant) pancreatitis to 71% for histopathologically moderate to severe pancreatitis. The wide overall range of sensitivities reported for cPLI (21% to 93%) is also seen with other markers for pancreatitis and reflects the inherent differences in the study design, the methodology, and the dog population used in each study. However, in the above mentioned studies, cPLI showed consistently the best performance (sensitivity and specificity) compared to other serum markers evaluated. For comparison, the sensitivity of various markers of pancreatitis reported in different studies ranges between 17% and 47% for serum cTLI concentration, 7% and 73% for serum amylase activity, and 13% and 69% for serum lipase activity.10,17,18,26,46 Based on the above mentioned studies, despite the fact that SNAP cPL and Spec cPL are the most sensitive serum markers for canine pancreatitis, false negative results do occur. This might be especially true for chronic pancreatitis. Based on some evidence from recent studies10,46,47 and the fact that histopathologic lesions associated with chronic pancreatitis such as pancreatic fibrosis and atrophy are not expected to be associated with leakage of pancreatic enzymes,36 the sensitivity of SNAP cPL and Spec cPL is likely lower for chronic pancreatitis than for acute pancreatitis. In one study of 14 dogs with chronic pancreatitis,46 the sensitivity of cPLI was only 26%. However, this study included several dogs that had exocrine pancreatic insufficiency (EPI) and while dogs with EPI may show an inflammatory infiltrate of the
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pancreas, this inflammation would unlikely be clinically relevant. In summary, exclusion of pancreatitis should ideally be based on a combination of clinical presentation and other diagnostic tests including routine laboratory tests (i.e., CBC, chemistry panel, urinalysis), imaging studies (i.e., ultrasonography, radiography), and potentially pancreatic cytology and/or histopathology. Similarly to its sensitivity, serum Spec cPL/cPLI is considered to have the highest specificity for pancreatitis compared to any other serum test currently available.7,9,18,26,36,47-51 Depending on the study, specificity ranges between 59% and 98%. However, as mentioned above, specificity is of limited importance for SNAP cPL because its recommended use is for the exclusion of pancreatitis (based on a normal result), while abnormal results do not allow for a definitive diagnosis of pancreatitis.
Clinical utility of SNAP fPL Similarly to the dog, the main use of SNAP fPL is to rule-out pancreatitis and not to diagnose pancreatitis. This recommendation has its basis on the high sensitivity of the SNAP fPL test (between 80% and 100% for severe acute pancreatitis), which means that a normal result makes a diagnosis of pancreatitis very unlikely. Therefore, sensitivity of this test is more important than its specificity and the following discussion will focus mainly on the sensitivity of this test and its negative predictive value. Abnormal SNAP fPL results can be seen in cats with Spec fPL concentrations in the gray zone or those with values that are consistent with pancreatitis. In the latter patients further testing using the quantitative Spec fPL is strongly recommended. Therefore, diagnosis of pancreatitis should not be based on the result of the SNAP fPL alone.
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In contrast to dogs, no studies in the peer reviewed literature have directly evaluated the sensitivity and specificity of the SNAP fPL for feline pancreatitis. However, as in dogs, the manufacturer indicates that the SNAP fPL test has a high level of agreement with the Spec fPL assay and therefore, the clinical performance characteristics of the SNAP fPL can be extrapolated from the performance characteristics of Spec fPL and fPLI assays with relative accuracy.43 In a study that has only been published in abstract form, SNAP fPL and Spec fPL were evaluated for the diagnosis of pancreatitis.52 In that study, the agreement between SNAP fPL and Spec fPL ranged between 90% and 98% (higher agreement for normal results) but no clear specific sensitivity and specificity was provided for either test. Studies in cats with both experimental and spontaneous pancreatitis have repeatedly shown that serum Spec fPL/PLI concentration is the most sensitive and specific serum marker for feline pancreatitis currently available.12,16,27,28,53-55 Similarly to dogs, it has proven particularly difficult to determine the exact sensitivity of fPLI, because this varies depending upon several factors, including the type of study, the criteria for pancreatitis used, and the severity of pancreatitis. In the only clinical study, that has only been reported in abstract form, 182 cats were studied and the sensitivity of serum Spec fPL concentration for acute pancreatitis was reported at 79%.54 As in dogs, a series of studies on the sensitivity of fPLI for feline pancreatitis based on using histopathology as the gold standard have been published, but it is more challenging to accurately interpret and determine the clinical significance of the results of those studies, because the diagnosis of pancreatitis was primarily based on histopathological criteria. Therefore, clinically healthy cats with histopathological lesions of the pancreas but clinically insignificant disease were also included in those studies.56 Three different studies have evaluated the sensitivity of Spec fPL/fPLI in cats. In one of those studies, the overall sensitivity
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of Spec fPL for the diagnosis of pancreatitis was 65%, using a cutoff value of 3.5 μg/dL, which corresponds to a normal result for SNAP.57 In the same study, the sensitivity for acute pancreatitis was 100%, while it was 58% for chronic pancreatitis. In an older study that included primarily cats with chronic pancreatitis, results were very similar: serum fPLI was found to be 100% sensitive for histopathologically moderate to severe pancreatitis, which was superior to the sensitivity of serum fTLI concentration (80%).53 In the same study, the sensitivity of fPLI was 54% for histopathologically mild pancreatitis, which was still considerably higher than that of serum fTLI (8%). In that study, the overall sensitivity of fPLI was 67%, while the one for fTLI was 28%. In the third and most recent of those studies, the sensitivity of Spec fPL (for either 3.5 μg/dL or 5.4 μg/dL as cut-off values) for the diagnosis of pancreatitis was 61%.56 Although categorization into acute and chronic pancreatitis was not available in that study, an activity index was calculated and the majority of cats included could be categorized as having mild chronic pancreatitis, as indicated by the very low activity indices. Therefore, the sensitivity of 61% likely corresponds to the sensitivity for chronic/mild pancreatitis in the two previously discussed studies (which were 58% and 54%, respectively). It is of note that in this last study, 95% of the cats examined had some degree of pancreatic inflammation, although cats were euthanized for a variety of reasons, and therefore the clinical relevance of those lesions remained unknown. Similarly to dogs, histopathologic lesions associated with chronic pancreatitis such as pancreatic fibrosis and atrophy are not expected to be associated with leakage of pancreatic enzymes, and therefore, the sensitivity of fPLI is lower for chronic pancreatitis than for acute pancreatitis as illustrated in the above studies. Therefore, as in dogs, false negative results cannot be excluded especially in cats with chronic or mild pancreatitis, and additional diagnostic modalities (especially imaging) should also be utilized.
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Similar to its sensitivity, the specificity of serum fPLI concentration for feline pancreatitis is very high, ranging between 63% and 100%.53,54,56,57 However, as mentioned above, specificity is of limited importance for SNAP fPL because its recommended use is for the exclusion of pancreatitis (based on a normal result), while abnormal results do not definitively confirm a diagnosis of pancreatitis. Although further studies are needed to evaluate the clinical performance characteristics of SNAP fPL in different groups of cats with a variety of diseases, it can currently be concluded that due to its high sensitivity especially for acute pancreatitis it is suitable for excluding this condition with relative confidence.
Conclusions The main use of SNAP cPL and SNAP fPL is to rule out pancreatitis in dogs or cats, respectively. Based on the available data to date, this can be done with 80% to 100% certainty for clinically apparent acute pancreatitis in both species. However, as with almost any test, falsenegative and false-positive results can occur, and therefore, diagnosis or exclusion of pancreatitis must additionally be based on clinical presentation in combination with a range of diagnostic tests including routine laboratory tests (i.e., CBC, chemistry panel, and urinalysis), imaging studies (i.e., ultrasonography and radiography), and potentially pancreatic cytology and/or histopathology.
Conflict of interest Dr. Steiner is a payed consultant of Idexx Laboratories that produces the SNAP fPL and SNAP cPL tests.
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immunoreactivity for the diagnosis of pancreatitis in cats. J Am Vet Med Assoc 2000;217:37-42. 29. Mansfield CS, Watson PJ, Jones BR. Specificity and sensitivity of serum canine pancreatic elastase-1 concentration in the diagnosis of pancreatitis. J Vet Diagn Invest 2011;23:691-697. 30. Westermarck E, Rimaila-Pärnänen E. Serum phospholipase A2 in canine acute pancreatitis. Acta Vet Scand 1983;24:477-487.
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31. Suchodolski JS, Collard JC, Steiner JM, et al. Development and validation of an enzymelinked immunosorbent assay for measurement of α1-proteinase inhibitor/trypsin complexes in canine sera. J Vet Intern Med 2001;15:311 [abstract]. 32. Suchodolski JS, Ruaux CG, Steiner JM, et al. Serum α1-proteinase inhibitor/trypsin complex as a marker for canine pancreatitis. J Vet Intern Med 2001;15:273 [abstract]. 33. Ruaux CG, Lee RP, Atwell RB. Detection and measurement of canine α-macroglobulins by enzyme immuno-assay. Res Vet Sci 1999;66:185-190. 34. Ruaux CG, Atwell RB. Levels of total α-macroglobulin and trypsin-like immunoreactivity are poor indicators of clinical severity in spontaneous canine acute pancreatitis. Res Vet Sci 1999;67:83-87. 35. Steiner JM, Berridge BR, Wojcieszyn J, et al. Cellular immunolocalization of gastric and pancreatic lipase in various tissues obtained from dogs. Am J Vet Res 2002;63:722-727. 36. Neilson-Carley SC, Robertson JE, Newman SJ, et al. Specificity of a canine pancreasspecific lipase assay for diagnosing pancreatitis in dogs without clinical or histologic evidence of the disease Am J Vet Res 2011;72 302-307. 37. Steiner JM, Williams DA. Purification of classical pancreatic lipase from dog pancreas. Biochimie 2002;84:1243-1251. 38. Steiner JM, Wilson BG, Williams DA. Purification and partial characterization of feline classical pancreatic lipase. Comp Biochem Physiol B Biochem Mol Biol 2003;134:151-159. 39. Steiner JM, Williams DA. Development and validation of a radioimmunoassay for the measurement of canine pancreatic lipase immunoreactivity in serum of dogs. Am J Vet Res 2003;64:1237-1241.
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40. Steiner JM, Teague SR, Williams DA. Development and analytic validation of an enzymelinked immunosorbent assay for the measurement of canine pancreatic lipase immunoreactivity in serum. Can J Vet Res 2003;67:175-182. 41. Steiner JM, Wilson BG, Williams DA. Development and analytical validation of a radiommunoassay for the measurement of feline pancreatic lipase immunoreactivity in serum. Can J Vet Res 2004;68:309-314. 42. Huth SP, Relford R, Steiner JM, et al. Analytical validation of an ELISA for measurement of canine pancreas-specific lipase. Vet Clin Pathol 2010;39:346-353. 43. Beall MJ, Cahill R, Pigeon K, et al. Performance validation and method comparison of an in-clinic enzyme-linked immunosorbent assay for the detection of canine pancreatic lipase. J Vet Diagn Invest 2011;23:115-119. 44. Haworth MD, Hosgood G, Swindells KL, et al. Diagnostic accuracy of the SNAP and Spec canine pancreatic lipase tests for pancreatitis in dogs presenting with clinical signs of acute abdominal disease. J Vet Emerg Crit Care 2014;24:135-143. 45. Kalenyak K, Schadow A, Burgener IA. Diagnosis of canine pancreatitis dependent on SNAP cPLTM and Spec cPLTM. Proceedings of the 22nd ECVIM Congress, 2012. 46. Watson PJ, Archer J, Roulois AJ, et al. Observational study of 14 cases of chronic pancreatitis in dogs. Vet Rec 2010;167:968-976. 47. Trivedi S, Marks SL, Kass PH, 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;25:1241-1247. 48. Steiner JM, Teague SR, Lees GE, et al. Stability of canine pancreatic lipase immunoreactivity concentration in serum samples and effects of long-term administration of
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prednisone to dogs on serum canine pancreatic lipase immunoreactivity concentrations. Am J Vet Res 2009;70:1001-1005. 49. Steiner JM, Finco DR, Gumminger SR, et al. Serum canine pancreatic lipase immunoreactivity (cPLI) in dogs with experimentally induced chronic renal failure. J Vet Intern Med 2001;15:311 [abstract]. 50. Hulsebosch SE, Palm CA, Segev G, et al. Evaluation of canine pancreas-specific lipase activity, lipase activity, and trypsin-like immunoreactivity in an experimental model of acute kidney injury in dogs. J Vet Intern Med 2016;30:192-199. 51. Mansfield CS, Anderson GA, O'Hara AJ. Association between canine pancreatic-specific lipase and histologic exocrine pancreatic inflammation in dogs: assessing specificity. J Vet Diagn Invest 2012;24:312-318. 52. Herrmann F, Hanisch F, Burgener IA. Diagnosis of feline pancreatitis dependent on SNAP fPLTM and Spec fPLTM. Proceedings of the 22nd ECVIM Congress, 2012. 53. Forman MA, Marks SL, De Cock HE, et al. Evaluation of serum feline pancreatic lipase immunoreactivity and helical computed tomography versus conventional testing for the diagnosis of feline pancreatitis. J Vet Intern Med 2004;18:807-815. 54. Forman MA, Shiroma J, Armstrong PJ, et al. Evaluation of feline pancreas-specific lipase (Spec fPLTM ) for the diagnosis of feline pancreatitis. J Vet Intern Med 2009:733-734 [abstract]. 55. Zavros NS, Rallis TS, Koutinas AF, et al. Clinical and laboratory investigation of experimental acute pancreatitis in the cat. Eur J Inflammation 2008;6:105-114. 56. Oppliger S, Hilbe M, Hartnack S, et al. Comparison of serum Spec fpl and 1,2-o-dilaurylrac-glycero-3-glutaric acid-(6'-methylresorufin) ester assay in 60 cats using standardized assessment of pancreatic histology. J Vet Intern Med 2016;30:764-770.
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57. Oppliger S, Hartnack S, Riond B, et al. Agreement of the serum Spec fPL and 1,2-odilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester lipase assay for the determination of serum lipase in cats with suspicion of pancreatitis. J Vet Intern Med 2013;27:1077-1082.
Figure legends Figure 1. SNAP cPL. This figure illustrates the interpretation of a SNAP cPL device. Panel (A) The patient spot (spot on the right) is lighter in color than the reference spot (spot on the left), which indicates a serum Spec cPL concentration within the reference interval and suggests that pancreatitis is unlikely. Panel (B) The patient spot (spot on the right) is darker in color than the reference spot (spot on the left), which indicates a serum Spec cPL concentration is above the upper limit of the reference interval and suggests that pancreatitis may be present. (From Small Animal Gastroenterology, 2008, J.M. Steiner (Ed.), Schlütersche, with permission).
Figure 2. Interpretation of SNAP PL results. This figure details the interpretation of SNAP PL devices. A negative SNAP PL indicates that the Spec PL is within the reference interval. A positive SNAP PL indicates that the Spec PL is either in the gray zone or consistent with pancreatitis. Therefore, an abnormal SNAP PL cannot be used to diagnose pancreatitis.
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PII: DOI: Reference:
S1938-9736(16)30031-9 http://dx.doi.org/10.1053/j.tcam.2016.10.005 TCAM266
To appear in: Topics in Companion Animal Medicine Cite this article as: Panagiotis G. Xenoulis and Jörg M. Steiner, SNAP Tests for Pancreatitis in Dogs and Cats: SNAP cPL and SNAP fPLSNAP tests for p a n c r e a t i t i s , Topics in Companion Animal Medicine, http://dx.doi.org/10.1053/j.tcam.2016.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
SNAP tests for pancreatitis in dogs and cats: SNAP cPL and SNAP fPL
Panagiotis G. Xenoulis1,2, Jörg M. Steiner3
1
Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, Karditsa, Greece
2
Animal Medical Center of Athens, Athens, Greece
3
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of
Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA
Short title: SNAP tests for pancreatitis
Key words: pancreatic lipase; pancreatitis; PLI; Spec cPL; Spec fPL
Correspondence: Dr. Panagiotis Xenoulis, Clinic of Medicine, Faculty of Veterinary Medicine, University of Thessaly, 224 Trikalon, Karditsa, 43100, Greece Email: [email protected], Tel. +30 24410 66085.
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Abstract A clinical diagnosis of pancreatitis in dogs and cats can be challenging. Several diagnostic modalities have been evaluated over the years for the diagnosis of canine and feline pancreatitis, but the majority of these modalities have been shown to be of limited clinical usefulness because of poor performance, limited availability, and/or because they are invasive. Assays for the measurement of pancreatic lipase immunoreactivity (Spec cPL in dogs and Spec fPL in cats) were first developed about 15 years ago, and studies have shown that they are currently the serum tests of choice for the evaluation of canine and feline patients, respectively, suspected of having pancreatitis. This is a direct consequence of their high specificity of detecting only pancreatic lipase and their sensitivity for pancreatitis when compared to other serum tests. SNAP cPL and SNAP fPL are in-clinic tests that have been developed based on the Spec cPL and Spec fPL assays. As with any other test, false positive and false negative results do occur with pancreatic lipase immunoreactivity assays and it is important to know the limitations of these assays.
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Introduction Accurate clinical diagnosis of pancreatitis in dogs and cats can be a big challenge in small animal practice. Several blood tests have been developed and evaluated for the diagnosis of canine and feline pancreatitis over the past decades, but most of them have been proven to be inadequate.1 In addition, several imaging modalities have been evaluated for the diagnosis of canine and feline pancreatitis, and with the exception of ultrasonography, none has been shown to be both valuable and practical for routine use for that purpose.1 During the last 15 years, pancreatic lipase immunoreactivity (PLI) assays and their companion SNAP PL (SNAP pancreatic lipase) tests have been developed, validated, and evaluated for the diagnosis of pancreatitis in dogs and cats, and have been proven to be useful.1,2 The present review will focus on SNAP cPL (SNAP canine PL) and SNAP fPL (SNAP feline PL) tests. A detailed review on the development and clinical use of canine and feline PLI assays has recently been published.2
Historical background: the need for development of new assays for canine and feline pancreatitis Assays for the measurement of serum amylase and lipase activities were initially developed more than 100 years ago, and were the first serum markers of pancreatic disease in humans (initially) and several animal species (much later).1,3,4 The application of these assays in small animal clinical practice did not start gaining popularity until the early 1960s, but at that time, information on the performance of those assays for the diagnosis of pancreatitis in dogs and cats was very limited.3 It was soon evident that the use of serum amylase and lipase activities as tests for the diagnosis of pancreatitis was associated with several inherent limitations.1 Several studies, both experimental and clinical, in dogs and cats showed that serum lipase and amylase 3
activities lack both sensitivity and specificity for the diagnosis of pancreatitis.1,5-12 Subsequently, it was shown that many tissues other than the pancreas (e.g., gastric mucosa, hepatic parenchyma, and many others) synthesize and release amylases and lipases.1,9,13-15 Also, generally speaking, catalytic assays cannot differentiate amylases and lipases according to their tissue of origin, which requires broadening the reference interval of these assays and thus not only accounts for the limited specificity (around 50% for both), but also the limited sensitivity for the measurement of serum amylase and lipase activities for pancreatitis in dogs.1,5-10,13,16 The reported sensitivity of serum amylase and lipase activities for spontaneous canine pancreatitis varies but is generally low (32% - 73% for lipase activity and 41% - 69% for amylase activity).1,10,17,18 In cats, both serum lipase and amylase activities do not appear to be of any clinical value in the diagnosis of spontaneous feline pancreatitis.1,12,19,20 Trypsin-like immunoreactivity (TLI) assays measure trypsinogen and trypsin in serum and were initially developed as more sensitive and specific markers of pancreatic disease in dogs and cats.21-24
30,31 33,34
These markers have become the gold standard for the diagnosis of
exocrine pancreatic insufficiency (EPI) in dogs22 and cats25 but studies have shown their sensitivity for the diagnosis of pancreatitis to be low for both dogs (36%-47%)10,26 and cats (28% - 64%).16,27,28 Several other diagnostic markers for pancreatitis have been developed and studied (e.g., serum concentrations of pancreatic elastase-1,29 phospholipase A2,30 trypsin-α1-antitrypsin complexes,31,32 and α2-macroglobulin,33,34 or plasma and urine concentrations of trypsinogen activation peptide (TAP)16,26), but none of these can currently be recommended for routine use in dogs and cats, either because their diagnostic performance has not been sufficiently evaluated clinically, or because they are associated with a limited sensitivity and/or specificity. Also, most of these assays are not routinely available.
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Pancreatic lipase As mentioned above, many tissues other than the pancreas (e.g., gastric mucosa, hepatic parenchyma, and others) synthesize and release lipases, which all share the same function (i.e., hydrolysis of triglycerides).1,9,13-15 This, in association with the limited ability (depending on the assay characteristics and conditions) of most traditional catalytic assays to differentiate lipases according to their tissue of origin results in low specificity and sensitivity of most serum lipase activity assays for pancreatitis in dogs and cats.1,5-14,17-20 Pancreatic lipase, which is of interest for the evaluation of the exocrine pancreas, is believed to be exclusively expressed by pancreatic acinar cells. This is supported by the findings of an immunolocalization study in dogs, that showed that canine pancreatic lipase was only expressed by pancreatic acinar cells among 38 different types of tissues.35 Another study in dogs with EPI showed a nearly total absence of serum cPLI concentration, further confirming that pancreatic lipase is exclusively of pancreatic origin.11 Similar studies have not been yet reported in cats. Although all lipases, including pancreatic lipase, share the same function, lipases of different cellular origin are encoded by different genes and have different amino acid sequences from one another and thus generate a distinct immunological response.14,15 Therefore, pancreatic lipase may function similarly to, but it is structurally different from other lipases. This means that assays that detect specific protein molecules based on their unique tertiary structure, such as immunoassays, should be able to specifically measure pancreatic lipase with no interference from other lipases. In addition, in contrast to catalytic lipase assays, immunoassays for pancreatic lipase would be species-specific because the pancreatic lipase amino acid sequence differs among species.
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PLI and Spec PL assays: the basis of SNAP PL tests SNAP PL tests are based on the technology, properties, and characteristics of the Spec PL assays, which were developed based on the original PLI assays. Therefore, in order to get a better understanding of the SNAP PL tests it is necessary to provide some basic information on the Spec PL assays. Pancreatic-lipase immunoreactivity assays are immunoassays developed to specifically quantify lipase of pancreatic origin based on its unique tertiary structure, while not detecting lipases of other tissue origin.11,14,15,35,36 Therefore, the PLI assays have inherent advantages over the traditional serum lipase activity assays that make them more suitable for specific evaluation of the exocrine pancreas in dogs and cats. For the development of the PLI assays, canine and feline pancreatic lipases were purified from canine and feline pancreas, respectively, following a sequence of purification experiments.37,38 Canine pancreatic lipase was found to be a low molecular weight protein of approximately 50.7 kDa37, while feline pancreatic lipase was found to have a molecular weight of 52.4 kDa using mass spectrometry.38 Following purification, antiserum directed against canine or feline pancreatic lipases was developed in rabbits by repeated inoculation of purified canine or feline pancreatic lipase, respectively.39-41 Purification of polyclonal antibodies was accomplished by affinity chromatography, and purified antibodies were used for the development of a radioimmunoassay (RIA)39 and a “sandwich” ELISA40 for canine PLI (cPLI) and an RIA for feline PLI (fPLI).41 All 3 assays were analytically validated, and were found to be sensitive, linear, accurate, precise, and reproducible for clinical application.39-41
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The originally developed cPLI and fPLI assays were in-house immunoassays that used small-scale manufacturing processes and therefore had a limited availability. In order for those assays to become suitable for commercial application and broad use, the originally developed assays had to be modified. The original cPLI ELISA and fPLI RIA assays were used as gold standards for the development of the new canine and feline assays, respectively. The new assays were named Spec cPL® (Specific Canine Pancreatic Lipase) and Spec fPL® (Specific Feline Pancreatic Lipase), and released for wide clinical use in 2007 and 2008, respectively, at which time they replaced the original assays. The main differences between the new and the old assays were as follows:39-42 a) the original PLI assays used purified pancreatic lipase whereas the Spec PL assays use a recombinant peptide as an antigen b) instead of using polyclonal antibodies produced in rabbits, the new assays use monoclonal antibodies secreted by cell lines that were produced in Balb/C mice; c) the original cPLI assay used a horseradish peroxidase (HRP) detection system that was added to the ELISA plate as a separate step, while the Spec PL assays use a pre-labeled anti-pancreatic lipase HRP conjugate allowing for direct detection. In a recent validation study, the Spec cPL assay was reported to be precise and reproducible for clinical application.42 The comparison of the two canine assays (the original cPLI and the Spec cPL) showed general agreement but also illustrated that a positive bias existed, with approximately 50% higher concentrations for Spec cPL compared to the original cPLI assay.42 This bias was accommodated by adjustments of decision limits, which were also evaluated and confirmed clinically.18,42 The reference interval for the Spec cPL assay is 0-200 µg/L, with concentrations above 400 µg/L being considered consistent with pancreatitis. Concentrations between 200 µg/L and 400 µg/L are considered in the “gray zone”.
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In contrast to the Spec cPL assay, details on the development and validation of the Spec fPL assay have not yet been published in the peer reviewed literature. Based on the manufacturer, the assay is precise and reproducible for clinical application, and has a good agreement with the original fPLI RIA. The reference interval for the Spec fPL assay is 0-3.5 µg/L and concentrations above 5.3 µg/L are considered consistent with pancreatitis. As in the dog, a “gray zone” exists for Spec fPL and corresponds to concentrations between 3.5 µg/L and 5.3 µg/L.
Development and validation of SNAP cPL® and SNAP fPL® assays In clinical practice, the time required to obtain results using the Spec cPL and Spec fPL is at least 24 hours, and might be considerably longer if weekends or holidays are included. The same is true for countries where the Spec cPL and Spec fPL are not available. This turnaround time, although generally acceptable in many situations, is unsuitable for the diagnosis of acute and especially severe acute pancreatitis that would potentially benefit from an early diagnosis and management. Therefore, rapid, in-clinic, semi-quantitative, visually-read tests (SNAP tests) for the estimation of canine and feline pancreatic lipase immunoreactivity in serum were developed and are based on the same ELISA technology and monoclonal antibodies as the quantitative reference methods (Spec PL).43 The SNAP tests incorporate a reference spot that corresponds to the upper limit of the reference interval for each species (200 µg/L for the dog and 3.5 µg/L for the cat), and the color intensity of the sample spot is visually compared to the reference spot (Figure 1).43 Therefore, the results of this rapid assay are considered either normal (less intense than the reference spot) or abnormal (equally to or more intense than the reference spot). In the latter case, the actual
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pancreatic lipase immunoreactivity concentration may be in the “gray zone” (200 µg/L to 400 µg/L for Spec cPL or 3.6 µg/L to 5.3 µg/L for Spec fPL) or consistent with a diagnosis of pancreatitis (>400 µg/L for Spec cPL or >5.3 µg/L for Spec fPL) (Figure 2).43 This differentiation cannot be determined with certainty, however, using the in-clinic assay and further testing using the quantitative reference method is recommended. A recent validation study showed agreement of close to 100% between the SNAP cPL and the reference Spec cPL method for samples in the reference interval, with a slightly lower agreement (about 90%) for samples above the upper limit of the reference interval.43 Samples that are close to the upper limit of the reference interval (200 µg/L) are more likely to be incorrectly classified.43 The SNAP cPL assay was found to be precise and reproducible and therefore suitable for clinical use.43 Although studies on the validation of the SNAP fPL have not been reported in the literature yet, the manufacturer indicates that in an in-house study that included 80 cats this test had an agreement of 89 - 93% for samples with results within the reference interval and a 92 - 100% agreement for samples with results with Spec fPL concentrations above the upper limit of the reference interval. Therefore, similarly to the SNAP cPL test, this test appears to be suitable for routine clinical use.
Clinical performance characteristics of SNAP cPL The main use of SNAP cPL is to rule-out pancreatitis rather than diagnose it. This recommendation has its basis on the high sensitivity of the SNAP cPL test (between 80% and 95% for clinical acute pancreatitis), which means that a normal result makes diagnosis of pancreatitis very unlikely. Therefore, sensitivity of this test is more important than its specificity and the following discussion will focus mainly on the sensitivity of this test and its negative
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predictive value. Abnormal SNAP cPL results can be seen in dogs with Spec cPL concentrations in the gray zone or those with cPLI concentrations consistent with pancreatitis and further testing using the quantitative Spec cPL assay would be necessary in such cases. Therefore, diagnosis of pancreatitis cannot be based on the result of the SNAP cPL alone. Two clinical studies have been published in the peer reviewed literature that have both evaluated and compared the diagnostic performance of Spec cPL and SNAP cPL. The first of those studies included 84 dogs, 57 of which had a clinical presentation indicative of acute pancreatitis.18 In the absence of a diagnostic test with perfect accuracy, traditional referencebased methods for evaluating test sensitivity and specificity were considered by the authors to yield biased estimates. Therefore, latent class analysis was used to determine unbiased estimates of diagnostic sensitivity and specificity for the tests evaluated. Dogs were categorized in groups based on the probability of having pancreatitis after a panel of 4 internists reviewed each dog’s complete medical record, laboratory findings, and abdominal ultrasound findings. SNAP cPL was found to have a sensitivity that ranged between 91% and 94%.18 In the same study, the sensitivity of the Spec cPL assay (cutoff value of 200 μg/L) was very similar (86 - 94%).18 The conclusion of that study was that SNAP cPL and Spec cPL have a higher sensitivity for diagnosing clinical acute pancreatitis than does measurement of serum amylase or lipase activity and that SNAP cPL and Spec cPL have a good negative predictive value when there is low prevalence of disease. The second study included 38 dogs that presented with acute abdominal disease, 11 of which were diagnosed as having acute pancreatitis based on history, physical examination findings, and the results of complete blood count, biochemical analysis, and abdominal ultrasonography performed by a veterinary radiologist.44 The SNAP cPL test was found to have a
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sensitivity of 82%, while the sensitivity of Spec cPL was 70%. As in the previous study, there was a good overall agreement between SNAP cPL and Spec cPL (≥ 200 μg/L) for the entire cohort (κ = 0.78).44 In the same study, it was found that the SNAP cPL test can give up to 40% false positive results for pancreatitis, which underlines the recommendation that a diagnosis of pancreatitis should not be based on the results of the SNAP cPL alone.44 A third study that has only been reported in abstract form to date also evaluated SNAP cPL and Spec cPL for the diagnosis of pancreatitis.45 This study included 114 dogs with a clinical suspicion of pancreatitis. In this study, SNAP cPL and Spec cPL showed an agreement of 88% for dogs suspected to have pancreatitis. However, sensitivity and specificity of the SNAP cPL test were not specifically reported in that abstract. With the exception of the 3 above-mentioned studies, no other studies have directly evaluated the sensitivity of SNAP cPL for pancreatitis. However, several other studies have evaluated Spec cPL (and/or the original cPLI) for the diagnosis of pancreatitis as these assays have been available for much longer compared to the SNAP cPL test. As mentioned above, several studies have established that the SNAP cPL test has a high level of agreement with the Spec cPL assay,18,43 and therefore, the clinical performance characteristics of the Spec cPL and cPLI assays can provide a relatively accurate estimation of the performance characteristics of the SNAP cPL test. A series of studies10,46,47 on the sensitivity of cPLI for canine pancreatitis based on histopathology as a gold standard have been published. However, it is more challenging to accurately interpret and determine the clinical significance of the results of these studies, because the diagnosis of pancreatitis was primarily based on histopathological criteria. Therefore, clinically healthy dogs with histopathological lesions of the pancreas but clinically insignificant
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disease were also included in those studies.10,47 It is of note that in the most recent of those studies,47 in which 70 dogs euthanized for a variety of reasons were examined, 63 dogs (90%) had histopathologic evidence of pancreatitis of which 56 (89%) had only mild disease. In addition, two of those studies enrolled primarily47 or exclusively46 dogs with chronic pancreatitis. In the above mentioned 3 necropsy studies,10,46,47 the sensitivity of Spec cPL/cPLI ranged from 21% for mild (potentially clinically insignificant) pancreatitis to 71% for histopathologically moderate to severe pancreatitis. The wide overall range of sensitivities reported for cPLI (21% to 93%) is also seen with other markers for pancreatitis and reflects the inherent differences in the study design, the methodology, and the dog population used in each study. However, in the above mentioned studies, cPLI showed consistently the best performance (sensitivity and specificity) compared to other serum markers evaluated. For comparison, the sensitivity of various markers of pancreatitis reported in different studies ranges between 17% and 47% for serum cTLI concentration, 7% and 73% for serum amylase activity, and 13% and 69% for serum lipase activity.10,17,18,26,46 Based on the above mentioned studies, despite the fact that SNAP cPL and Spec cPL are the most sensitive serum markers for canine pancreatitis, false negative results do occur. This might be especially true for chronic pancreatitis. Based on some evidence from recent studies10,46,47 and the fact that histopathologic lesions associated with chronic pancreatitis such as pancreatic fibrosis and atrophy are not expected to be associated with leakage of pancreatic enzymes,36 the sensitivity of SNAP cPL and Spec cPL is likely lower for chronic pancreatitis than for acute pancreatitis. In one study of 14 dogs with chronic pancreatitis,46 the sensitivity of cPLI was only 26%. However, this study included several dogs that had exocrine pancreatic insufficiency (EPI) and while dogs with EPI may show an inflammatory infiltrate of the
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pancreas, this inflammation would unlikely be clinically relevant. In summary, exclusion of pancreatitis should ideally be based on a combination of clinical presentation and other diagnostic tests including routine laboratory tests (i.e., CBC, chemistry panel, urinalysis), imaging studies (i.e., ultrasonography, radiography), and potentially pancreatic cytology and/or histopathology. Similarly to its sensitivity, serum Spec cPL/cPLI is considered to have the highest specificity for pancreatitis compared to any other serum test currently available.7,9,18,26,36,47-51 Depending on the study, specificity ranges between 59% and 98%. However, as mentioned above, specificity is of limited importance for SNAP cPL because its recommended use is for the exclusion of pancreatitis (based on a normal result), while abnormal results do not allow for a definitive diagnosis of pancreatitis.
Clinical utility of SNAP fPL Similarly to the dog, the main use of SNAP fPL is to rule-out pancreatitis and not to diagnose pancreatitis. This recommendation has its basis on the high sensitivity of the SNAP fPL test (between 80% and 100% for severe acute pancreatitis), which means that a normal result makes a diagnosis of pancreatitis very unlikely. Therefore, sensitivity of this test is more important than its specificity and the following discussion will focus mainly on the sensitivity of this test and its negative predictive value. Abnormal SNAP fPL results can be seen in cats with Spec fPL concentrations in the gray zone or those with values that are consistent with pancreatitis. In the latter patients further testing using the quantitative Spec fPL is strongly recommended. Therefore, diagnosis of pancreatitis should not be based on the result of the SNAP fPL alone.
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In contrast to dogs, no studies in the peer reviewed literature have directly evaluated the sensitivity and specificity of the SNAP fPL for feline pancreatitis. However, as in dogs, the manufacturer indicates that the SNAP fPL test has a high level of agreement with the Spec fPL assay and therefore, the clinical performance characteristics of the SNAP fPL can be extrapolated from the performance characteristics of Spec fPL and fPLI assays with relative accuracy.43 In a study that has only been published in abstract form, SNAP fPL and Spec fPL were evaluated for the diagnosis of pancreatitis.52 In that study, the agreement between SNAP fPL and Spec fPL ranged between 90% and 98% (higher agreement for normal results) but no clear specific sensitivity and specificity was provided for either test. Studies in cats with both experimental and spontaneous pancreatitis have repeatedly shown that serum Spec fPL/PLI concentration is the most sensitive and specific serum marker for feline pancreatitis currently available.12,16,27,28,53-55 Similarly to dogs, it has proven particularly difficult to determine the exact sensitivity of fPLI, because this varies depending upon several factors, including the type of study, the criteria for pancreatitis used, and the severity of pancreatitis. In the only clinical study, that has only been reported in abstract form, 182 cats were studied and the sensitivity of serum Spec fPL concentration for acute pancreatitis was reported at 79%.54 As in dogs, a series of studies on the sensitivity of fPLI for feline pancreatitis based on using histopathology as the gold standard have been published, but it is more challenging to accurately interpret and determine the clinical significance of the results of those studies, because the diagnosis of pancreatitis was primarily based on histopathological criteria. Therefore, clinically healthy cats with histopathological lesions of the pancreas but clinically insignificant disease were also included in those studies.56 Three different studies have evaluated the sensitivity of Spec fPL/fPLI in cats. In one of those studies, the overall sensitivity
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of Spec fPL for the diagnosis of pancreatitis was 65%, using a cutoff value of 3.5 μg/dL, which corresponds to a normal result for SNAP.57 In the same study, the sensitivity for acute pancreatitis was 100%, while it was 58% for chronic pancreatitis. In an older study that included primarily cats with chronic pancreatitis, results were very similar: serum fPLI was found to be 100% sensitive for histopathologically moderate to severe pancreatitis, which was superior to the sensitivity of serum fTLI concentration (80%).53 In the same study, the sensitivity of fPLI was 54% for histopathologically mild pancreatitis, which was still considerably higher than that of serum fTLI (8%). In that study, the overall sensitivity of fPLI was 67%, while the one for fTLI was 28%. In the third and most recent of those studies, the sensitivity of Spec fPL (for either 3.5 μg/dL or 5.4 μg/dL as cut-off values) for the diagnosis of pancreatitis was 61%.56 Although categorization into acute and chronic pancreatitis was not available in that study, an activity index was calculated and the majority of cats included could be categorized as having mild chronic pancreatitis, as indicated by the very low activity indices. Therefore, the sensitivity of 61% likely corresponds to the sensitivity for chronic/mild pancreatitis in the two previously discussed studies (which were 58% and 54%, respectively). It is of note that in this last study, 95% of the cats examined had some degree of pancreatic inflammation, although cats were euthanized for a variety of reasons, and therefore the clinical relevance of those lesions remained unknown. Similarly to dogs, histopathologic lesions associated with chronic pancreatitis such as pancreatic fibrosis and atrophy are not expected to be associated with leakage of pancreatic enzymes, and therefore, the sensitivity of fPLI is lower for chronic pancreatitis than for acute pancreatitis as illustrated in the above studies. Therefore, as in dogs, false negative results cannot be excluded especially in cats with chronic or mild pancreatitis, and additional diagnostic modalities (especially imaging) should also be utilized.
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Similar to its sensitivity, the specificity of serum fPLI concentration for feline pancreatitis is very high, ranging between 63% and 100%.53,54,56,57 However, as mentioned above, specificity is of limited importance for SNAP fPL because its recommended use is for the exclusion of pancreatitis (based on a normal result), while abnormal results do not definitively confirm a diagnosis of pancreatitis. Although further studies are needed to evaluate the clinical performance characteristics of SNAP fPL in different groups of cats with a variety of diseases, it can currently be concluded that due to its high sensitivity especially for acute pancreatitis it is suitable for excluding this condition with relative confidence.
Conclusions The main use of SNAP cPL and SNAP fPL is to rule out pancreatitis in dogs or cats, respectively. Based on the available data to date, this can be done with 80% to 100% certainty for clinically apparent acute pancreatitis in both species. However, as with almost any test, falsenegative and false-positive results can occur, and therefore, diagnosis or exclusion of pancreatitis must additionally be based on clinical presentation in combination with a range of diagnostic tests including routine laboratory tests (i.e., CBC, chemistry panel, and urinalysis), imaging studies (i.e., ultrasonography and radiography), and potentially pancreatic cytology and/or histopathology.
Conflict of interest Dr. Steiner is a payed consultant of Idexx Laboratories that produces the SNAP fPL and SNAP cPL tests.
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31. Suchodolski JS, Collard JC, Steiner JM, et al. Development and validation of an enzymelinked immunosorbent assay for measurement of α1-proteinase inhibitor/trypsin complexes in canine sera. J Vet Intern Med 2001;15:311 [abstract]. 32. Suchodolski JS, Ruaux CG, Steiner JM, et al. Serum α1-proteinase inhibitor/trypsin complex as a marker for canine pancreatitis. J Vet Intern Med 2001;15:273 [abstract]. 33. Ruaux CG, Lee RP, Atwell RB. Detection and measurement of canine α-macroglobulins by enzyme immuno-assay. Res Vet Sci 1999;66:185-190. 34. Ruaux CG, Atwell RB. Levels of total α-macroglobulin and trypsin-like immunoreactivity are poor indicators of clinical severity in spontaneous canine acute pancreatitis. Res Vet Sci 1999;67:83-87. 35. Steiner JM, Berridge BR, Wojcieszyn J, et al. Cellular immunolocalization of gastric and pancreatic lipase in various tissues obtained from dogs. Am J Vet Res 2002;63:722-727. 36. Neilson-Carley SC, Robertson JE, Newman SJ, et al. Specificity of a canine pancreasspecific lipase assay for diagnosing pancreatitis in dogs without clinical or histologic evidence of the disease Am J Vet Res 2011;72 302-307. 37. Steiner JM, Williams DA. Purification of classical pancreatic lipase from dog pancreas. Biochimie 2002;84:1243-1251. 38. Steiner JM, Wilson BG, Williams DA. Purification and partial characterization of feline classical pancreatic lipase. Comp Biochem Physiol B Biochem Mol Biol 2003;134:151-159. 39. Steiner JM, Williams DA. Development and validation of a radioimmunoassay for the measurement of canine pancreatic lipase immunoreactivity in serum of dogs. Am J Vet Res 2003;64:1237-1241.
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57. Oppliger S, Hartnack S, Riond B, et al. Agreement of the serum Spec fPL and 1,2-odilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) ester lipase assay for the determination of serum lipase in cats with suspicion of pancreatitis. J Vet Intern Med 2013;27:1077-1082.
Figure legends Figure 1. SNAP cPL. This figure illustrates the interpretation of a SNAP cPL device. Panel (A) The patient spot (spot on the right) is lighter in color than the reference spot (spot on the left), which indicates a serum Spec cPL concentration within the reference interval and suggests that pancreatitis is unlikely. Panel (B) The patient spot (spot on the right) is darker in color than the reference spot (spot on the left), which indicates a serum Spec cPL concentration is above the upper limit of the reference interval and suggests that pancreatitis may be present. (From Small Animal Gastroenterology, 2008, J.M. Steiner (Ed.), Schlütersche, with permission).
Figure 2. Interpretation of SNAP PL results. This figure details the interpretation of SNAP PL devices. A negative SNAP PL indicates that the Spec PL is within the reference interval. A positive SNAP PL indicates that the Spec PL is either in the gray zone or consistent with pancreatitis. Therefore, an abnormal SNAP PL cannot be used to diagnose pancreatitis.
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