Diagnostic Laboratory Services- von Willebrand's disease in dogs
Introduction
von Willebrand's Disease (vWD) is a condition that may result in an animal to bleed. This bleeding may be of a varying tendency and severity between affected individuals. In dogs it may be an acquired condition associated with an underlying disease, but is most commonly seen as an inherited condition. Acquired forms of the disease are often susceptible to treatment of the underlying disease, the concentration of von Willebrand Factor (vWF) returning to normal in many cases.
For those animals with the inherited condition there is no possibility of a cure. When aware of a decrease in vWF concentration, informed breeders, owners and their veterinarians can be aware of the possibility of complications due to accident, injury and/or surgery and propagation of this trait can be reduced or eliminated by informed breeding decisions.
von Willebrand's Disease results in a reduced quantity and/or function of von Willebrand's factor (vWF), a glycoprotein necessary for normal platelet function in the initial phase of haemostasis (blood clotting). It is caused by the inheritance of mutated genes that code for the production of von Willebrand Factor, these mutated genes are also referred to as the von Willebrand's Disease genes. von Willebrand's Disease is inherited as an autosomal recessive trait. This means an animal (male or female) with two mutated vWF genes will be affected with the disease. An animal who has inherited 1 mutated gene will be a carrier of von Willebrand's Disease. If 2 carrier animals are mated, there is a probability that half of the resulting offspring will be carriers, 25% will be affected with the disease, and 25% will be normal. There is variable expression of the vWF trait, so some affected animals may bleed severely while other affected animals may hardly show any bleeding at all. Therefore, we can not reliably predict which dogs with low plasma vWF concentrations will show impairment of haemostasis.
Breeds of dogs with a high prevalence of the vWD gene (15-80%) include the Basset Hound; standard and miniature Dachshund; Doberman Pinscher; German Shepherd; Golden Retriever; Keeshond; standard and toy Manchester Terrier; miniature Schnauzer; Pembroke Welsh Corgi; miniature and standard Poodle; Rottweiler; Scottish Terrier and Shetland Sheepdog. Von Willebrand’s Disease has been documented in other breeds and/or cross bred dogs, but the prevalence of the vWD gene is lower than 15% or insufficient data has been accumulated in order to determine prevalence.
Clinical signs of bleeding that are typical of the decrease in platelet function seen with vWD include bleeding from the gums, urinary system, epistaxis (nose bleed) and/or gastrointestinal bleeding, with or without diarrhoea. Petechiae (small haemorrhages) on the gums or any mucosal surfaces may be apparent. Animals with vWD may be prone to prolonged bleeding at any site of injury, trauma or surgery. Haemorrhage into body cavities, joints or large extravasations are more suggestive of other coagulation factor deficiencies, but do not rule out the presence of vWD.
Not all animals with decreased levels of vWF exhibit increased tendency to bleed. Obtaining a buccal mucosal bleeding time may be of benefit in determining if an individual with decreased vWF concentration is likely to exhibit clinical signs of bleeding, with or without trauma. Buccal mucosal bleeding time may be used to help formulate classification and recommendations for breeding (See Interpretation section). Surgicutt buccal mucosal bleeding time devices are available from Diagnostic Laboratory Services at the cost of £10.00. Instructions and expected bleeding times are supplied with the device.
Method of Testing
Animal Health Trust refers samples to Cornell University, New York for testing by the Enzyme-Linked Immunosorbent Assay (ELISA) method for vWF determination in blood plasma. It compares patient vWF antigen concentration to that of a known canine standard.
Animal Health Trust offer a genetic test for von Willebrand’s disease, which carried out at our Newmarket site. This test is only available for Irish Red & White Setters. For more information contact our Canine Genetics Group on 08700 509144, or visit the AHT website: www.aht.org.uk.
In healthy animals the vWF concentration may be used to predict genetic status with regard to vWD. Heterozygous carriers of vWD usually are identified as having less than half the normal concentration of plasma vWF. The homozygous condition results in production of little or no vWF protein.
There is a borderline level of vWF concentration (50-69%) at which accurate prediction of vWD status is not possible. It may be the result of normal genotype or heterozygous carrier of the vWD gene. Retesting and/or breeding only to higher testing mates is recommended and puppies should be checked.
The rate of misclassification of genetic status by the ELISA test has been reported to be less than 5%. However, misclassification of genotype by false positive or false negative result is always possible. Retesting of an additional, freshly drawn sample is recommended in any animal with a result suspected of being falsely positive or negative. In addition, analysis of samples from the parents of progeny affected with vWD is recommended, regardless of their previous status - they may have been misclassified as free from vWD. Review of sample collection, handling and testing conditions, as well as animal condition, possible health problems and recent medications also should be considered.
Ageing of Samples
Ageing of the sample may effect vWF levels, as may repeat freezing and thawing.
Our studies of sample ageing at room temperature over 1 - 12 days indicate a decline in vWF concentration of up to 37%. Decreases of up to 18% may occur with only 2 days at room temperature with progressive, but smaller decreases occurring up to 7 days. After 7 days there appears to be more variation in levels with possible plateau effect.
Studies of sample stability while frozen at -20oC for 1 week indicate no significant decrease in vWF concentration during this time.
This is important since thawing of plasma samples during prolonged transport may cause decreases that would change the interpretation of the test results.
Indications for Testing
The vWF test is indicated in dogs in the following circumstances:
- Prior to breeding. Animals known to be at risk for vWD based on breed, clinical signs and/or history should be tested. In a healthy animal, vWF concentration may be used to predict genetic status.
- In conjunction with other tests in animals with clinical signs of bleeding, conditions that may predispose to acquired vWD or other causes of bleeding.
Conditions Contraindicating Submission of Samples for vWF Screening
The concentration von Willebrand Factor may be influenced by a variety of hormones, medications and inflammatory conditions. This may result in unpredictable fluctuations of vWF concentrations. Samples for vWF testing should not be collected if the following apply.
- Vaccination, medication including steroids) or resolution of systemic illness within the last 2 weeks
- Recent blood transfusion, within the last week.
- Recent surgery, as vWF may participate as an acute-phase reactant protein and increase with inflammation or stress. This test should be scheduled prior to surgery if there is concern about increased risk associated with breed, clinical signs or history.
- Pregnancy, oestrus (heat) or lactation. An anoestrus sample is recommended for female dogs.
- Suspected endocrine disease that has not been evaluated. Hypothyroidism may aggravate decreases in vWF. Other endocrine conditions may influence vWF concentrations. Concurrent evaluations of endocrine and vWF status may be of benefit in some individuals, based on clinical signs, presentation and history.
- We also recommend that puppies are tested at least 2 weeks after their ‘last’ vaccination.
Sample Collection & Handling
Careful collection and handling are critical in obtaining an accurate result. With improper sample collection there may be early activation of haemostasis prior to the visual detection of a clot, which tends to produce falsely low vWF concentration results.
- Sodium citrate is the only suitable anti-coagulant for von Willebrand Factor testing.
- Use only plastic syringes and tubes, as glass tends to cause activation of haemostasis resulting in the formation of a clot.
- The correct ratio of citrate anticoagulant (3.8% Tri-sodium citrate) to blood is important, so the tube(s) should not be under or over-filled.
- Usually collection of a minimum volume of 2.0 ml of blood is requested to ensure that an adequate volume of plasma is obtained.
- Remove the needle from the syringe and gently expel blood down the side of a plastic sodium citrate tube. Do not squirt blood through the needle as this may result in the destruction of red blood cells.
- The sample must be mixed gently but thoroughly immediately after collection, to prevent activation of haemostasis, that may cause a falsely low of von Willebrand Factor result.
- If a clot is present, the sample is not suitable for submission and should be recollected.
- Prompt centrifugation and separation of plasma from the blood cells is requested if possible. Since prolonged contact of cells and plasma may result in haemolysis, or transcellular shifts of constituents that may interfere with the testing or reduce the volume of plasma available for harvesting.
- If your sodium citrate sample tube will not fit into your centrifuge, transfer the blood to an appropriately sized, clean, plain plastic tube (no anti-coagulant) for centrifugation. The sample should be centrifuged at 2000-3000 rpm for 10 minutes or using the settings that you usually use to separate blood in your centrifuge.
- If excessive haemolysis is present, in the plasma after centrifugation the sample may not be suitable for testing and recollection is advised.
- Carefully pipette plasma, taking care that erythrocytes are not aspirated. Use a plastic pipette and transfer plasma to a clean, plain, plastic tube that does not contain anticoagulant.
- Please label the tube with the animal and owner name, date and contents (citrated plasma).
- For the von Willebrand Factor Antigen test we recommend that the plasma be frozen. Samples kept at room temperature have been shown to give lower results compared to samples that have been stored refrigerated or frozen.
- We recommend that the sample is sent to the laboratory by as soon as possible, ie overnight delivery (by special delivery post or courier). Ice packs should be included with the sample packaging for vWF testing to ensure that the sample remains cold.
- Please do not send samples on Friday since the weekend may delay delivery. In this case keep the sample frozen and post on Monday.
When samples are received for von Willebrand Factor testing, they are stored frozen until the next test. Samples are sent to Cornell University, USA by courier every other Wednesday with results available by Monday morning.
