LABOKLIN aktuell 2016 – LABOKLIN Europe

Pancreatitis – Pancreatic insufficiency: What do PLI, TLI, Vitamin B12 and Co tell us?

Laboklin — Sun, 12 Jun 2016 12:47:14 +0000

The pancreas is a very important digestive organ in dogs and cats and consists of an exocrine and endocrine portion. The exocrine pancreas is a serous gland that secretes enzymes that are essential for the digestion of all relevant types of nutrients. Anatomically, it is made up of the acini and the duct system. The enzymes are produced by the secretory cells in the acini and are stored in granules.

1. Pancreatitis

A pancreatitis is a very painful inflammation of the pancreas, caused by early activation and release of proteolytic enzymes from the acinar cells. The result is local and systemic destruction of tissues by the pancreatic enzymes themselves and by inflammatory mediators that are released during this process. Middle aged dogs and cats are most commonly affected. Miniature Schnauzers, Yorkshire Terriers, and Poodles are prone to pancreatitides. Siamese cats appear to be more commonly affected than other cat breeds. (Fig. 1 + Fig. 2)

Pancreatitis has long been known as a disease in companion animals. Inflammatory changes were first noted more or less accidentally during dog and cat necropsies. This led to the hypothesis that pancreatitides are much more common in dogs and cats than previously supposed. Numerous researchers have since contributed to a better understanding of this disease. However, although we now know that diseases of the exocrine pancreas, especially pancreatitis, are much more common in dogs and cats than originally supposed, clinical diagnosis and treatment have not become easier. The principal signs of disease are very unspecific and include anorexia, vomitus, abdominal pain, weakness, diarrhoea, (icterus in cats). Not all signs are present in all cases and in very mild cases no clinical signs may be detected. In cats, the signs are even less clear than in dogs. For this reason, pancreatitis should always be included in the list of differential diagnoses in cases of gastrointestinal disease. (Fig. 3 + Fig. 4)

2. Pancreatitis – Diagnosis/Laboratory Diagnosis

Blood count and clinical chemistries usually only show unspecific changes that may be related to vomiting and diarrhoea. The changes do not help in reaching a diagnosis, but can help evaluate the health status of the patient.

2.1. Amylase/Lipase

These are not pancreas specific enzymes and are also found in the intestine, the muscles, the salivary glands, and the liver. The sensitivity of lipase is about 55% in dogs. A 3-fold lipase increase is relatively specific in dogs, but lipase is not always elevated in cases of pancreatitis. In cats, lipase is even less specific than in dogs. Both enzymes may be increased in liver and kidney disease, in cases with neoplasia or following treatment with glucocorticoids and diabetic ketoacidosis.

2.2. TLI (Trypsin-like Immunoreactivity)

This assay measures trypsin and its precursor trypsinogen. Trypsinogen is synthesized exclusively in the pancreas. However, only 30-60% of the dogs and cats with pancreatitis have measurable increases in this value. This is probably due to the very short half-life of the enzyme. Feeding and kidney disease also lead to increased values. The TLI is a better diagnostic marker for exocrine pancreas insufficiencies.

2.3. PLI (Pancreatic Lipase Immunoreactivity)

Pancreas lipase is synthesized exclusively in the acinar cells of the pancreas. Physiologically, only small amounts of pancreatic lipase are found in the peripheral circulation. If acinar cells are destroyed during the course of a pancreatitis, increased amounts are released into the circulation. The degree of increase correlates with the degree of inflammation and destruction of the pancreas. The pancreatic immunoreactivity is determined by structural characteristics specific for pancreatic lipase. This is a significant difference from other assays which determine the lipase activity. The activity, however, is identical for all lipases, independent of the tissue from which they originated. Specific tests for the quantification of feline (fPLI) and canine (cPLI) pancreatic lipase have been developed. The specificity is around 71% in dogs with a sensitivity of 93%. In cats, the assay is 91% specific and 67% sensitive. The determination of PLI is not influenced by enzyme substitution and neither haemolysis nor lipaemia affect the determination. PLI is very stable. The determination of PLI in serum from dogs and cats is currently considered the most reliable non-invasive test for the diagnosis of pancreatitis.

3. Exocrine Pancreatic Insufficiency (EPI)

In cases of EPI, the synthesis and secretion of pancreatic enzymes are insufficient, leading to maldigestion and malabsorption. Clinical signs occur when the functional tissue has been reduced to 10-15%. An EPI can manifest itself in weight loss despite normal or increased appetite. Affected animals often suffer from chronic diarrhoea and the coat may appear shaggy and dull. The faeces of affected dogs is often unformed, very voluminous and may be yellowish and fatty and contain undigested food. An EPI can be congenital or acquired. German Shepherds and Collies can have a genetic defect that causes pancreatic acinar atrophy (PAA). Affected dogs develop clinical signs at a very young age and can become severely cachectic. EPI is very often a result of chronic recurring pacreatitides that have not been recognized and treated. In these cases it is a disease of middle aged or old dogs and cats. In rare cases, pancreatic neoplasia can lead to EPI. If the EPI is a consequence of a chronic recurring pancreatitis, diabetes mellitus can develop as a secondary complication. In almost all cases, EPI is associated with vitamin B12 deficiency. This is due to the fact that intrinsic factor, which is necessary for the resorption of vitamin B12 in the ileum, is synthesized in the pancreas. Necessary buffer systems and antimicrobial substances, which are also products of the exocrine pancreas, are also lacking. Inflammatory bowel disease (IBD) therefore often develops during the course of EPI. In cats, EPI is rare and is usually the result of a chronic pancreatitis or an obstruction of glandular discharge (adenocarcinoma). (Fig. 5)

4. EPI Diagnosis

4.1. Clinical Chemistry and Blood Count

Often unremarkable.

4.2. TLI (Trypsin-like Immunoreactivity)

As mentioned above for pancreatitis, TLI determines trypsin and its inactive precursor trypsinogen in the serum of dogs and cats in species specific assays. It is the test of choice for the diagnosis of exocrine pancreatic insufficiency. The sensitivity and specificity of TLI are near 100% in dogs. The sensitivity in cats is unknown, but the specificity is between 85 and 100%. EPI is characterized by a decreased TLI serum concentration, although a single low TLI value without corresponding clinical signs is not diagnostic of EPI and should be confirmed after a few weeks. Only if the serum TLI concentration is persistently reduced is there proof of an EPI.

Careful: the animal must fast (8, better 12 hours) before sampling. Food intake leads to artificially increased values. False high values are also found in conjunction with kidney insufficiency and in severely cachectic dogs.

4.3. Pancreatic Elastase

Pancreatic elastase is an endoprotease that is resistant to digestion which is exclusively synthesized in the acinar cells of the pancreas. It can currently only be measured in faecal material from dogs. There is no corresponding assay available for cats. Pancreatic elastase levels are significantly reduced in exocrine pancreatic insufficiencies, but can also be reduced in healthy dogs, so that a positive result should be confirmed by a follow-up determination of TLI (specificity 50%). It should be noted that there is a “dilution effect” in patients with diarrhoea. In these cases, the test should be repeated and the serum TLI should be determined.

4.4. Vitamin B12 (Cobalamin)/Folic Acid

These are both water soluble vitamins of the B-complex. They cannot be synthesized by the body and must be ingested from the diet. Commercial foods general contain sufficient amounts of both vitamins, making food based deficiencies improbable unless an animal is fed an exclusively vegetarian diet. Vitamin B12 is bound to food proteins and must be cleaved from these with the help of pancreatic enzymes, and then bound to intrinsic factor, which is also synthesized in the pancreas, in order to be resorbed in the ileum.

In cases of exocrine pancreatic insufficiency, insufficient intrinsic factor is synthesized, reducing the resorption of vitamin B12 in the ileum. Many intestinal bacteria also digest vitamin B12, which increases the deficiency. Folic acid is absorbed via specific receptors in the small intestine. Damage of those receptors during the course of a disease can lead to a deficiency. A folic acid surplus can also occur in cases of overgrowth or miscolonisation of the small intestine, since intestinal bacteria are able to synthesize folic acid. Since EPI often leads to shifts in the intestinal flora, changes in folic acid and vitamin B12 concentration can be helpful for diagnostics. These interdependencies demonstrate that an EPI can easily cause vitamin B12 deficiency and increased serum folic acid levels. Checking both vitamins is therefore recommended for all patients with gastrointestinal signs and/or suspected EPI.

Pancreatitis – Pancreatic insufficiency

Pathology Bulletin

Laboklin — Thu, 12 May 2016 13:29:32 +0000

Histology

Microscopic examination of tissue sections following formalin fixation (10%) and embedding in paraffin is used to diagnose many different lesions including tumours, lesions in organs or in the skin, or inflammation and infection.

All slides are routinely stained with H.E. (hematoxylin and eosin). Special staining techniques are also available (e.g. for the detection of fungi, mycobacteria, mast cells).

Please remember the following when submitting samples:

• Send a sufficient number of representative samples
• Avoid artefacts during extraction: electrocoagulation, tearing, crushing, and autolysis are detrimental for the analysis
• Ideal sample size: 0.4-1.0 cm (enough tissue to evaluate, small enough for complete fixation, some variation is possible due to extraction technique or case. The evaluation of resection margins requires e.g. submission of all of the tissue (possibly including tumour bed biopsies).
• Formalin fixation is absolutely necessary! Relationship of the volume of fixative: tissue size at least 1:10, better 1:20.
• Do not use other fixatives (e.g. alcohol). It is better to submit the material unfixed (1 work day is generally not a problem).
• At temperatures below zero, the addition of a small amount of alcohol prevents freezing.
• A case history is necessary (on the filled out submission form): e.g. species and breed, age, treatment, clinical signs, extraction site, clinical question, differential diagnoses.
• For an appropriately submitted, fixed sample, the lab report will be ready on the work day after the sample arrives in the laboratory (Monday-Friday). It can take longer for unfixed or hard samples that have to be decalcified or if special stains are necessary.

Cytology

Microscopic examination of air dried smear preparations is used to evaluate cells (inflammatory cells, tumour cells, cells from organs).

Cytology smears are routinely stained according to Pappenheim. It is also possible to evaluate submissions that have been stained using Diff-Quick®. If necessary, special stains are used (e.g. for fungi, mycobacteria, copper).

Please remember the following when submitting samples:

• Possible techniques: smear following aspiration or puncture, impression smears, cytobrush, tissue swabs rolled onto slides
• In order to avoid autolysis (esp. urine, CSF), the cytology slides should be prepared in the practice
• Avoid artefacts, especially caused by thick smears or the use of too much pressure
• Always air dry slides, never use heat fixation or cover slips
• Submit puncture material/fluid along with the slides
• Centrifuge fluids with a low amount of cellular material (clear fluids) and use the sediment to prepare the smear (indicate “sediment” on the submission form)
• Bloody fluids should be submitted in EDTA tubes in order to prevent coagulation
• A case history is crucial (please fill out the submission form)
• A cytology report is usually ready on the same day the sample arrives in the laboratory (Monday-Friday)

The charges for histology and cytology depend on the number of problems. For example, examination of multiple skin biopsies for a dermatological case costs only the basic price. If, for example, a tumour is also submitted, the charge would be for two examinations, or twice as much.

Shipping and packaging materials (submission forms, formalin containers, envelopes, containers for slides) are available free of charge at any time.

If additional testing may be of interest, material that can be used for such tests should be included (e.g. swabs for bacterial culture).

Immunohistochemisty:

Goal: Antigen detection in tissue sections from paraffin blocks using tagged antibodies following histological evaluation. Immunohistochemistry is used in addition to histology in the diagnosis of tumous and for the detection of infectious agents in tissues. This type of test cannot be used for the diagnosis of autoimmune diseases.

Determining the cellular origin in tumour diagnostics, e.g.:

• Cytokeratin (epithelial marker)
• Vimentin (mesenchymal marker)
• Melan A (melanocytes)
• CD3/CD79a (T-/B-cells)

Determining expression profiles for prognostic/therapeutic purposes, e.g.:

• C-Kit, Ki-67 (mast cell tumours in dogs)
• Cox-2 (bladder and intestinal tumours)

Detection of infectious agents in tissues, e.g.:

• Feline herpesvirus, parvovirus, FIP

Clonality testing/PARR:

The PARR test (PCR for antigen receptor rearrangement), a molecular test to deter mine the clonality of lymphocytes in dogs and cats, has become very important. Detection of a monoclonal proliferation of B or T-cells indicates with a high probability that these are the source of a lymphoma.

The great advantage of the technique is that the suspicious cells (derived from a paraffin block or from stained or unstained slides) can be used directly without the need for renewed sampling. All tissues/fluids in which sufficient numbers of lymphocytes are found can be used for testing.

Pathology Bulletin