Source: Problems in Veterinary Medicine - Vol. 4, No. 4, December 1992, TRANSFUSION MEDICINE pp. 636-646.
Von Willebrand’s disease (vWD) is the most common canine inherited bleeding disorder.1-3 It comprises a group of bleeding diseases; variations exist in genetic transmission, clinical severity, and diagnostic laboratory findings. Differences in underlying pathogenetic mechanisms cause this variable expression in affected breeds of dog. In addition to breed-specific variants of inherited vWD, there are apparent “acquired” forms of vWD in many breeds of dog.3,4,7,8 Because of the clinical variability of canine vWD, treatment must be tailored to meet each affected dog’s specific needs.
The common feature in all forms of vWD is reduction in the amount of functional von Willebrand’s factor (vWF), a large plasma glycoprotein 1,2,9,10 that is produced in endothelial cells and has a critical role in supporting platelet adhesion to sites of small vessel injury. The net result of interactions between damaged endothelium, platelets, and vWF is localized formation of a platelet plug. These interactions are called the primary phase of hemostasis. In larger vessels, activation of the clotting cascade results in formation of more permanent fibrin clots around the framework of platelet plugs. Interactions of the primary phase of hemostasis are distinct from enzymatic reactions of the clotting cascade, and vWF does not participate as a factor or cofactor in the coagulation cascade.10,11
In plasma, vWF molecules are bound together in series like links in a chain. The length of these chains, known as multimers, is variable. It appears that the longest chains, consisting of the largest multimers, are most active in supporting platelet adhesion. 10,11
Abnormal hemostasis in dogs with vWD results from quantitative and qualitative defects of vWF protein.1,2 The most severe hemostatic defect is seen in dogs having no detectable plasma vWF or having reduced concentrations of structurally abnormal vWF. Milder forms of vWD occur in dogs having reduced concentrations of a structurally normal protein.1-6
DIAGNOSIS OF VON WILLEBRAND’S DISEASE
In Vivo Tests
Bleeding time tests are in vivo measures of primary hemostasis and are useful screening tests for the identification of dogs with vWD.12-14 However, abnormal bleeding time is not specific for vWD. Thrombocytopenia, inherited thrombopathies, acquired platelet dysfunction, hemophilia, and anemia are causes of long bleeding times reported in human beings.15,16 Two relatively simple techniques have been described for measuring bleeding time in dogs: cuticle bleeding time (CBT) and buccal mucosa bleeding time (BMBT).12,13,17 These tests are performed by producing a standard wound; incising the toenail cuticle or buccal mucosa of upper lip; and recording the time from incision until cessation of blood flow from the wound. Cuticle bleeding time is sensitive to deficiencies of coagulation factors and defects of primary hemostasis.17 Buccal mucosa bleeding time appears to be more specific for primary hemostatic defects.12 Normal CBT for dogs is approximately 4-6 minutes, and normal BMBT is approximately 2-4 minutes.12-14,17 Dogs with severe cases of vWD have marked prolongation of bleeding time (more than 20 minutes) and steady bleeding from incised wounds. Dogs with less severe cases have bleeding times that are variably prolonged beyond normal range, with finite end points.12,14 Bleeding times should be considered rough estimates of in vivo hemostasis in patients with vWD. Clinical severity of bleeding, especially after surgical procedure or trauma, is dependent on vWF function and other factors, such as the anatomic site, type of insult, and amount of tissue injured.
In Vitro Tests
Definitive diagnosis of canine vWD requires specific measurement of canine vWF protein. 1-3 Routine hemostatic screening tests, such as platelet count, coagulation assays (activated clotting time, activated partial thromboplastin time, prothrombin time, thrombin time, and fibrinogen concentration) and titers of fibrin split products are not sensitive to deficiencies or dysfunction of vWF.1 Although vWF circulates in plasma bound to coagulation Factor VIII, measurements of Factor VIII are neither specific nor sensitive enough to substitute for direct measurement of vWF.3,9,10
Canine vWF is antigenically, functionally, and structurally distinct from human vWF.18-22 Samples should be submitted to laboratories that have specifically validated canine vWF assays. Sample quality is critical for accurate measurement of vWF. Plasma samples for routine vWF assays should be prepared from blood collected in citrate or ethylenediamin-etetraacetic acid (EDTA) anticoagulant. Serum samples and plasma containing clots or severe hemolysis are invalid for measurement of canine vWF.3,23
The von Willebrand factor antigen (vWF:Ag ) assay is routinely performed as a quantitative measure of canine vWF.18-19 Antibodies directed against antigens present on canine vWF protein are used to determine concentration of vWF in a sample. Concentration is reported as percentage of vWF:Ag or units/deciliter vWF:Ag compared with a control standard plasma. Test results should be interpreted relative to each laboratory’s established normal range of canine plasma vWF:Ag. Results from different laboratories are not directly comparable because plasma standards, assay techniques, and normal ranges vary among laboratories.1-3,23 Measurements of plasma vWF:Ag concentration are used as predictors of genetic status for the vWD trait and for the diagnosis of vWD in dogs with bleeding diatheses.1-4,24
The following ranges, using an ELISA assay technique for measurement of canine plasma vWF:Ag, have been established at the Comparative Hematology Laboratory 18,24: normal range, 70-180% vWF:Ag; borderline range, 50-69% vWF:Ag; and abnormal range, 0-49% vWF:Ag.
Dogs that test in the normal range are considered to not have the vWD trait and are considered to be a low risk for expressing or transmitting the disease.
Dogs that test in the borderline range can not be accurately classified as carriers or as clear of vWD on the basis of the measurement. This is an overlap region of plasma percentage vWF:Ag for which some dogs are genetically clear and some are carriers of vWD. It is unusual for dogs with more than 50% vWF:Ag to express the vWD trait and to exhibit a clinically significant bleeding diathesis.
Dogs that test in the abnormal range are considered carriers of vWD and are at risk for transmitting an abnormal vWF gene to offspring. Carriers that have no plasma vWF (0% vWF:Ag) invariably express a bleeding tendency. However, severity of bleeding diathesis for carriers having low plasma vWF is not necessarily proportional to a reduction in plasma vWF:Ag concentration. Inheritance and expression pattern of vWD within each breed and the presence or absence of concurrent disorders may influence severity of bleeding tendency in individual carriers.2-6
Functional vWF assays, known as cofactor assays, are performed by combining normal platelets with an agglutinating reagent that is dependent on vWF for activity.9,10 Comparisons are made between the agglutination response obtained using patient plasma as the source of vWF versus dilutions of normal canine plasma. The antibiotic ristocetin is used routinely in human vWF cofactor assays, but it is difficult to use in canine test systems.20 Botrocetin, a protein derived from snake venom, is more commonly used in canine vWF cofactor assays.21,24 Botrocetin cofactor (BCf) assays are semiquantitative measures of vWF and are dependent on concentration and function of vWF in test plasma. BCf assays are relatively imprecise compared with vWF:Ag assays and are somewhat difficult to standardize. Cofactor assays usually are performed when evaluating patients with a bleeding diathesis; they are used as a screening test to identify dogs with low plasma vWF.23 However, predictions of clinical severity of vWD that are based solely on abnormal BCf activity are not accurate. In general, the results of BCf assays provide no more clinical information than do results of vWF:Ag assays.
Structural vWF assays measure vWF multimer size distribution.10,11,22 Multimer assays are technically complex and are not routinely performed when screening for canine vWD. In research laboratories measurements of plasma vWF concentration and multimer size are used, making it possible or enabling differention of certain types of vWD in affected breeds of dogs. These canine variants are comparable to three broad categories used to describe vWD in humans.9,10 Type I vWD is characterized by low concentration of structurally normal vWF protein. This form is the most common in human and canine medicine, and severity of bleeding diathesis is variable in affected individuals.1-4,9 Type II vWD is characterized by low concentration of abnormal vWF (i.e., specifically deficient of the largest vWF multimers). Most affected individuals have a moderate to severe bleeding diathesis.2-4,9 Type III vWD is characterized by virtual absence of vWF in quantitative or qualitative assays. Type III vWD invariably is a severe bleeding diathesis.1-4,9 Table 1 presents a summary of characteristics of dogs with Types I, II, and III vWD.
CLINICAL FEATURES OF CANINE VON WILLEBRAND’S DISEASE
History and Signs
Typical signs of vWD include bleeding from mucosal surfaces (e.g., epistaxis, hematuria, melena) and excessive bleeding and bruising from sites of trauma or surgical procedure.1-4 Specific signs in a particular affected dog are dependent on a number of variables. Puppies with severe forms of vWD often have noticeable gingival bleeding when deciduous teeth are shed; local or systemic treatment to control hemorrhage is required.4-6 The urinary tract and oronasal cavities are rich in local fibrinolysins. Surgery or minor injury to these tissues may lead to severe bleeding in dogs with vWD, whereas skin incisions or superficial wounds heal without complication. Abnormal hemostasis may not be clinically apparent in dogs with mild cases until a critical anatomic site, such as the central nervous system is injured. Compared with normal dogs, dogs with vWD may exhibit exaggerated signs of bleeding if they develop concurrent disorders that affect hemostasis. Commonly encountered disorders include thrombocytopenia, drug administration, hypothyroidism, liver disease, and uremia.1-4
Table 1. Characterization of Canine von Willebrand’s Disease
Type I Low Normal Variable Doberman, Corgi, Sheltie,
German Shepherd, Akita, Poodle*
Type II Low Abnormal(large Severe German Shorthaired Pointer
Type III Undetectable Undetectectable Severe Scottie, Sheltie, Chesapeake
Retriever, German Wirehaired
* Type I vWD has been identified in most common purebreeds.
Rarely is vWD the sole cause of petechiae, hemarthroses, or hematoma formation. Thrombocytopenia is by far the most common cause of petechiae in dogs. Acquired or inherited coagulation factor deficiencies are the usual cause of bleeding into body cavities, subcutaneous tissues, or muscles.1,2
Prevalence of Von Willebrand’s Disease
vWD has been identified in more than 50 different breeds of dog.1,3,24 Genetic screening programs for the vWD trait have been established in many of these breeds.3,4,24 Data for the six breeds with the largest testing programs for a 3-year period (1988-1990) is presented in Table 2. The information was compiled through the Comparative Hematology Laboratory’s nationwide vWD testing program.
Table 2. Prevalence of the von Willebrand’s Disease Trait in Six Breeds (1988-1990)*
Breed Average Number of Dogs
Average Percentage of Dogs
Tested in Carrier Range+
Doberman Pinscher 1625 75
Pembroke Welsh Corgi 181 43
Shetland Sheepdog 824 35
Scottish Terrier 343 30
Golden Retriever 644 30
Poodle (Standard and Miniature) 476 30
* Based on data from the Comparative Hematology Laboratory.
+ Carrier range defined as plasma vWF:Ag<50%
Within these six breeds and in other breeds with less extensive testing programs, there are variations in the proportion of carriers expressing the vWD trait and the severity of their bleeding diathesis.1-4 Doberman Pinschers, Scottish Terriers, and Shetland Sheepdogs are the breeds most often affected with clinical signs of vWD.4 Affected lines or severe forms of vWD also have been identified in Akitas, Old English Sheepdogs, German Shorthaired Pointers, Schnauzers, Rough Coasted Collies, and Basset Hounds.1-4 All purebred, as well as mixed breed, dogs have some apparent risk for expressing vWD. Within the last few years, sporadic cases of severe forms of vWD have been diagnosed in Pomeranians, Fox Terriers, Alaskan Huskies, and German Wirehaired Pointers (Comparative Hematology Laboratory, unpublished data).
Inheritance and Expression Patterns
vWD is inherited as an autosomal trait; males and females have equal risk for transmitting the defect or expressing a bleeding diathesis.1-4,9 Heterozygous carriers of vWD usually are identified as having less than half the normal concentration of plasma vWF (i.e., < 50% vWF:Ag), and homozygous carriers produce virtually no vWF protein (0% vWF:Ag). 1-4,24
In most breeds, the vWD trait is incompletely dominant. Heterozygotes that have a penetrant form of the defect express a bleeding diathesis, whereas other heterozygotes that have an impenetrant form have no symptoms.2-4 Family studies and pedigree analysis of vWD-affected Doberman Pinschers, Pembroke Welsh Corgis, German Shepherds, and most other breeds have shown variable expression of a bleeding diathesis in heterozygotes; this expression is compatible with incomplete penetrance of the vWD defect.3,24, An abnormal vWF gene transmitted from either parent is sufficient to produce affected offspring. In these breeds, homozygosity for the vWD trait apparently is lethal. All affected dogs have low, but measurable, plasma vWF and normal vWF multimer structure; these characteristics are typical of Type I vWD.2-4,24 In vitro assays of plasma vWF can not reliably differentiate carriers that will express a bleeding diathesis from those that have low plasma vWF without clinically relevant impairment of hemostasis.1-4 Additional evidence of expression of vWD can be obtained from evaluation of history and in vivo bleeding time.1-4,23
A simple recessive pattern of expression is found in Scottish Terriers with vWD.1-4 In this breed, dogs with the disease are homozygous for the vWD trait, have no detectable plasma vWF, and invariably express a severe bleeding diathesis. These findings are typical of Type III vWD.2-4,24 In this form, both parents must transmit an abnormal gene to produce affected offspring. Heterozygous carriers have low plasma vWF concentration, but they do not experience abnormal hemostasis.2-4 An apparent recessive inheritance and expression pattern also has been reported in Chesapeake Bay Retrievers.5
Shetland Sheepdogs are believed to have an incompletely dominant inheritance pattern of vWD, with variable but relatively mild bleeding diathesis seen in some heterozygotes.3,6 In recent years, a severe form of Type III vWD has been identified in this breed; affected dogs have no detectable plasma vWF.6 Family studies of these dogs have shown them to be offspring of two carrier parents; these findings are compatible with a homozygous or doubly heterozygous state.
German Shorthaired Pointers with vWD express a severe bleeding diathesis.2,4,25 The inheritance pattern in this breed is not well defined. Affected dogs have been the offspring of two clinically normal parents, findings that are compatible with incompletely dominant or recessive inheritance. Results of in vitro vWF assays have shown low plasma concentration of vWF and relative deficiency of large vWF multimers; such findings are typical of Type II vWD.2,25 this form of vWD has not been identified in any breed other than the German Shorthaired Pointer.
Acquired von Willebrand’s Disease
A hemostatic defect, with signs typical of vWD, has been described in adult purebred and mixed breed dogs that had no prior or familial history of abnormal hemorrhage.1,7,8,26 Dogs usually had excessive mucosal or postoperative hemorrhage. Diagnostic evaluation of these dogs reveals abnormal bleeding time and low plasma vWF concentration; normal platelet count and normal clotting times were found in coagulation assays. Signs of a concurrent disease process, usually an endocrine disorder, often were present.1,7 Thyroid insufficiency is considered the most common associated clinical disorder, but hormonal changes associated with estrus, parturition, and Addison’s disease have been found.4,7,23,26 Correction of the underlying disorder is followed by resolution of bleeding diathesis within a few days. In some, but not all cases, plasma vWF concentration increases to within normal range.26 A similar hemorrhagic syndrome that is responsive to thyroid hormone supplementation has been described in hypothyroid humans.27,28
The pathogenetic mechanism of acquired vWD is not well defined in humans or dogs. Reversible impairment of hemostasis may be secondary to changes in intracellular or subendothelial vWF and in plasma vWF. The acquired defect may be attributable, in part, to abnormalities of platelet function or hemostatic proteins other than vWF. In vitro assays of plasma vWF in patients with acquired vWD reveal low concentration of structurally normal vWF protein; such findings are typical of patients with inherited Type I vWD.7,26-28 Until more specific diagnostic tests are found, dogs suspected of having acquired vWD should be evaluated to rule out other acquired bleeding disorders, including thrombocytopenia, metabolic disease or drugs causing platelet dysfunction, and defects of the coagulation cascade or fibrinolytic pathways.
MANAGEMENT OF DOGS WITH VON WILLEBRAND’S DISEASE
Successful management of dogs with vWD includes definitive diagnosis and specific treatment of affected dogs during a bleeding crisis and identification of carriers that express an abnormal bleeding diathesis before they undergo surgical procedures.
Initial Patient Assessment
Plasma samples for vWF assay should be drawn before extensive drug or transfusion therapy. Common causes of bleeding diatheses in dogs, including thrombocytopenia, coagulation factor deficiency, and acquired platelet dysfunction, should be ruled out. Slide estimates of platelet number and activated clotting time are useful quick assessment tests.1,29 Bleeding caused by thrombocytopenia usually is not expected if examination of a stained blood film under oil immersion reveals at least 7-10 platelets per field. Normal range of canine activated clotting time is 60-120 seconds. Abnormalities detected in quick assessment tests can be studied more closely with more definitive tests, including platelet count, coagulation screening assays, fibrinogen concentration, and fibrin (ogen) degradation product titers.29 Thorough drug history and metabolic profile usually reveal conditions that affect platelet function, especially therapy with nonsteroidal anti-inflammatory drugs, uremia, or hyperproteinemia.29,30 In some cases, additional diagnostic workup may be indicated to identify underlying endocrinopathies that influence expression of vWD.1-3,23
When measurement of plasma vWF is used as a prediction of genetic status for the vWD trait, the most accurate results are obtained during physiologic “quiet” times.23,24 vWF is a hemostatic protein and also acts as an acute-phase reactant protein.10,23 Concentration of plasma vWF tends to fluctuate in association with systemic infectious, inflammatory and neoplastic disorders, and hormonal changes that accompany heat cycles, pregnancy, and lactation. The magnitude and direction of change from baseline vWF concentration is unpredictable, and samples for vWF:Ag (used as genetic predictor) should be drawn at least 2 weeks after vaccination, medication, or resolution of systemic disease.3
Measurement of in vivo bleeding time is useful in the initial workup of dogs with bleeding diathesis (after platelet and clotting disorders have been ruled out).1,2,23,29 Prolongation of bleeding time (CBT or BMBT) is compatible with a diagnosis of vWD. Preoperative measurement of bleeding time also can be performed for Doberman Pinschers and other breeds that have a high prevalence of the vWD trait. In general, dogs having clinically significant expression of the trait have measurable prolongation of bleeding time. However, predictions of intraoperative or postoperative bleeding complications should not be based solely on bleeding time. In each case, the type of surgical procedure and overall status of the patient’s other hemostatic mechanisms should be considered.
Nontransfusion Supportive Care
Dogs with severe forms of vWD have a lifelong risk of experiencing serious bleeding episodes. For these dogs, avoidance of invasive diagnostic procedures or elective surgical procedures is recommended. If dogs with vWD undergo surgical procedure, close postoperative monitoring is required. Excessive bleeding may not be obvious during a procedure but may occur during anesthetic recovery. In some dogs with vWD, rebleeding from incised tissues has been observed as long as 24 hours after surgical procedure.
It is good practice in dogs with vWD to avoid drug or vaccine adminstration that might compromise normal platelet function.1,30 Vaccination with certain modified live vaccines causes transient thrombocytopenia within 1 week of administration. Surgery or other stressful situations should be eliminated or minimized until platelet numbers rebound, usually within 2 weeks.1,3 Nonsteroidal anti-inflammatory drugs (aspirin, phenylbutazone, actaminophen, and ibuprofen) impair platelet function.30 Administration of these drugs can cause or exacerbate a bleeding episode in dogs with vWD. In general, the use of anti-inflammatory doses or corticosteroids (dexamethasone, prednisone, and prednisolone) is preferable to any of the nonsteroidal anti-inflammatory drugs.
Other drugs that are associated with thrombocytopenia or impaired platelet function include most chemotherapeutic agents, sulfa and sulfa trimethoprim combination drugs, chloramphenicol, quinidine, phenobarbital, heparin, and estrogen.30 Use of these drugs should be avoided. If they are administered, the patient’s clinical status and platelet count should be monitored closely.
Local wound treatment at the site of surgical or traumatic tissue injury often reduces blood loss in patients with vWD.1,3,23 Useful procedures for improving local hemostasis include electrocautery, ligation of small subcutaneous vessels, multilayer closure of incisions, and application of pressure wraps. Orthopedic procedures and surgical procedures that involve mucosal surfaces represent severe hemostatic challenges. Bleeding from wounds in the oral cavity, including tooth extraction sites, often is encountered and may be difficult to control. Absorbable sponges can be packed into gingival defects and kept in place with sutures. Topical tissue adhesive is useful for controlling hemorrhage from small mucosal defects. Adhesives are most effective if applied to a wound after bleeding is controlled with direct pressure and the tissues are dry.
Hormonal therapy is indicated for certain patients if differential diagnosis includes acquired vWD.1-4,23 Many of the same breeds with a high prevalence of the vWD trait also are at increased risk of developing thyroid insufficiency.31-33 For adult dogs that develop a bleeding tendency in association with hypothyroidism, supplementation with thyroid hormone often prevents or reverses significant bleeding episodes. Thyroid hormone should be given at a replacement dose of 0.02 mg/kg twice a day. The dose should be adjusted to maintain postpill T4 concentration near the high end of the normal canine range.7 Dogs that do not respond should be carefully reassessed to identify a focal source of hemorrhage (including occult neoplasia) or systemic bleeding diathesis other than vWD.
Table 3. Blood Products and Dosages to Supply von Willebrand Factor
Product Dose Frequency
Fresh* whole blood 12-25 mg/kg q 24 hr
Fresh* plasma 6-10 ml/kg q 8-12 hr
Fresh frozen plasma+ 6-10 ml/kg q 8-12 hr
Plasma cryoprecipitate 1 unito/10 kg q 6-8 hr
* Fresh indicates that the blood product was transfused within 6 hours of collection.
+ Fresh frozen plasma separated from whole blood and frozen within 6 hours of collection.
o 1 unit is the amount of cryoprecipitate produced from 150 ml of fresh frozen plasma.
Desmopressin acetate (DDAVP, deamino 8-D-arginine vasopressin) is a synthetic vasopressin analog that is used as pharmacologic treatment for some forms of vWD in humans.9,10 The drug is believed to stimulate release of intracellular stores of vWF, which results in increased concentrations of plasma vWF.34 It is not effective in patients who are genetically incapable of producing vWF or in those who produce certain dysfunctional forms of the protein. The duration of response to desmopressin is transient (hours), and there is a poor response to repeated doses given within a 24-hour period.9,10,34 A summary of trials evaluating the effect of desmopressin in healthy dogs with normal vWF concentration and Doberman Pinschers with vWD has been reported.23 In general, desmopressin caused an increase in plasma vWF concentration in healthy dogs. The magnitude of response was variable and was less than that seen in humans. Affected Dobermans had only nonrelevant increases of plasma vWF concentration after desmopressin administration. However, shortening of BMBT has been reported in some Dobermans with vWD one-half hour after subcutaneous administration of desmopressin at a dose of 1 ug/kg.35 Because of the unpredictable canine response to desmopressin, routine use of the agent in dogs with vWD is not recommended. Corrections of abnormal bleeding time should be demonstrated before desmopressin is used as preoperative prophylaxis. In addition, close intraoperative and postoperative monitoring is required to ensure adequate duration of response.
Transfusion to supply active vWF is needed to control hemorrhage in dogs with severe forms of vWD and dogs unresponsive to supportive care or hormonal therapy. Blood products that contain functional vWF include fresh whole blood, fresh plasma, fresh frozen plasma, and plasma cryoprecipitate.1,36-38 Table 3 presents dosage and frequency guidelines for transfusing these products.
Ideally, blood donors should have the same blood type and should have been found on crossmatch to be compatible donors for dogs with vWD that have received prior transfusions and dogs likely to receive multiple transfusions. Donor dogs negative for red blood cell antigens DEA 1.1, 1.2, and 7 usually are considered “universal” donors, those least likely to sensitize recipients to foreign antigens.1,37 Blood donors should be screened for the vWD trait to ensure that they have plasma vWF:Ag within the normal range.
Intravenous catheters for administering blood products to dogs with severe cases of vWD should be placed in a peripheral vein, rather than the jugular vein, to avoid perivascular hematoma or hemorrhage that might interfere with respiration. If intravenous catheterization is impossible, most red cells and plasma proteins transfused via the intraosseous route will gain access to the peripheral circulation.37 The intramedullary cavities of the femur, ilium, humerus, and tibia are potential sites of intraosseous transfusion.
Transfusions of blood products that contain red blood cells are indicated when patients with vWD have signs that are indicative of acute or chronic blood loss anemia. Stored whole blood and packed red blood cells do not contain relevant amounts of active vWF, but the plasma of fresh whole blood, transfused within 6 hours of collection, supplies some functional vWF.1,36 Transfusion of products that contain red blood cells, whether or not they contain active vWF, often stabilize the condition of dogs with vWD and allow time for nontransfusion supportive care to be initiated and take effect. However, repeated administration of these products puts the recipient at risk of adverse immunologic transfusion reactions and volume overload and does not supply sufficient vWF to improve hemostasis to a marked degree.1,36,37 For these reasons, dogs with vWD that have no clinical signs of anemia are best treated with blood components that more selectively supply vWF.
Plasma separated from whole blood and transfused within 6 hours of collection is considered fresh plasma (FP).1,36-38 Fresh frozen plasma (FFP) is produced by centrifuging whole blood within 6 hours of collection and separating and rapidly freezing the plasma supernatant. Storage of FFP at low temperatures (as low as -70o C) preserves the activity of hemostatic proteins, including vWF, for as long as 1 year.38 Transfusions of FP and FFP supply roughly an equivalent amount of active vWF (in half volume) as the whole blood from which they were prepared. In general, these products are transfused through 150 um blood filters at a rate of 6-10 ml/minute.1 Plasma can be administered prophylactically to patients with vWD within a few hours of invasive procedures; repeated transfusions (minimum interval, every 8 hours) can be given within a 24-hour period. However, there is some risk of volume overload with the administration of multiple plasma transfusions.23,36,37
Cryoprecipitate is a plasma concentrate prepared from FFP that is enriched in vWF, coagulation Factor VIII, fibrinogen, and fibronectin.1,36-38 The volume of cryoprecipitate that forms, when FFP is slowly thawed, is about one-tenth that of the initial plasma. One unit of cryoprecipitate (defined as that amount formed from approximately 150 ml plasma), is administered for each 10 kg of recipient body weight. Cryoprecipitates can be stored frozen at low temperatures for as long as 1 year.36-38 A major advantage of cryoprecipitate transfusion is the large amount of active vWF supplied in small volumes of infusate. Second or third doses of cryoprecipitate can be transfused in 1-2-hour intervals without causing volume overload.23 Maintenance transfusions (sometimes needed in dogs with severe cases of Type II or III vWD) can be given at 6 hour intervals. Cryoprecipitates also are convenient to transfuse preoperatively because the entire dose can be given during a period of 10-15 minutes. The dose can be repeated intraoperatively or postoperatively.
Transfusion of selected blood components, rather than whole blood, optimizes the benefit of transfusion and minimizes the risk of adverse reactions.36-38 Cryoprecipitate transfusions are the most reliable means of supplying active vWF to control hemorrhagic crises in dogs with vWD.1,23,37 Increased plasma vWF:Ag concentrations, in some cases as much as three to four times greater than pretreatment values, have been measured in dogs with vWD after cryoprecipitate transfusion (Comparative Hematology Laboratory, unpublished data). To improve efficacy of canine cryoprecipitates, some researchers recommend administration of desmopressin to donor dogs before blood collection.2,23 The expected increase in concentration or activity of vWF in products prepared from these donors or the specific beneficial response in a series of recipients have not been well described.
More widespread availability of blood components is expected as commercial companies and veterinary teaching hospitals develop canine donor programs and blood banking techniques. Controlled clinical trials are needed to determine the optimum treatment protocols for dogs with various forms of vWD. Clinical and laboratory characteristics of dogs with vWD and the specific hemostatic insult they encounter should be thoroughly defined in the comparisons of different treatment protocols. Objective measures of plasma vWF, bleeding time, and clinical response should be included in trials to clarify the effects of different transfusion and nontransfusion treatments.
Knowledge of the inheritance and expression pattern of vWD within different breeds of dog and the evaluation of clinical history, endocrine profile, plasma vWF, and bleeding time in individual dogs provide a good framework for choosing the appropriate treatment and effective management of canine vWD.
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26. Avgeris S, Lothrop CD, McDonald TP: Plasma von Willebrand factor concentration and thyroid function in dogs. J Am Vet Med Assoc 1989;196:921-924.
27. Dalton DG, Savidge GF, Matthews KB, et al: Hypothyroidism as a cause of acquired von Willebrand’s disease. Lancet 1987; 2:1007-1009.
28. Palareti G, Biagi C, Legnani C, et al: Association of reduced Factor VIII with imparired platelet reactivity to adrenalin and collagen after total thyroidectomy. Thromb Haemost 1989; 64:1053-1056.
29. Johnstone IB: Diagnostic approach to the bleeding patient, Current Veterinary Therapy X. In: Kirk RW, ed. Philadelphia: WB Saunders, 1989:436-442.
30. Catalfamo JM, Dodds WF: Hereditary and acquired thrombopathias. Vet Clin Noarth Am 1988; 18:185-193.
31. Beale, KM, and Halliwell, RE, Chen CL: Prevalence of antithyroglobulin antibodies detected by enzyme-linked immunosorbent assay of canine serum. J Am Vet Med Assoc 1989; 196:745-748.
32. Nachreiner RF, Refsal KR, Thaker EL, et al: Incidence of T3 and T4 autoantibodies in dogs using a sensitive binding assay (abstract). J Vet Intern Med 1990;4:114.
33. Chastain CB: Canine hypothroidism. J Am Vet Med Assoc 1982; 181:349-353.
34. Mannucci PM: Desmopression: A new nontransfusional form of treatment for congenital and acquired bleeding disorders. Blood 1988; 72:1449-1455.
35. Kraus KN, Turrentine MA, Jergens AE, Johnson GS: Effect of desmopressin acetate on bleeding times and plasma von Willebrand factor in Doberman pinscher dogs with von Willebrand’s disease. Vet Surg 1989; 18:103-109.
36. Turnwald GH, Pichler ME: Blood transfusion in dogs and cats. Part II: Administration, adverse effects and compent therapy. Compend Contin Educ Pract Vet 1985; 7:115-122.
37. Cotter S: Blood banking I and II. Proceedings ACVIM Sixth Annual Forum; Washington DC, 1988:45-50.
38. Authement JM, Wolfsheimer KJ, Catchings S: Canine blood component therapy: Product preparation, storage and administration. Journal of the American Animal Hospital Association 1987; 23:483-493.
Original Doc: vwd06.doc
Inherited And Acquired Von Willebrand’s Disease
W. Jean Dodds, DVM*, Sharon L. Raymond, BS,
and Marjory B. Brooks, DVM, Dip. ACVIM
Hemopet, Santa Monica, CA 90403 and Wadsworth Center
for Laboratories and Research, New York State Department
of Health, P.O. Box 509, Albany, NY 12201-0509
*Address to send correspondence: 938 Stanford Street, Santa Monica, CA 90403. Supported in part by NIH Research Grant HL09902 from the National Heart and Blood Institute, PHS/DHHS.
von Willebrand’s disease (vWD) in human beings and dogs can be either an inherited, congenital disorder present at birth and/or an acquired syndrome secondary to familial autoimmune thyroid disease. 1-7 In humans, acquired vWD also develops occasionally in association with cancer, systemic autoimmune disorders and other causes.3,4 Of the 57 dog breeds recognized to date to have vWD, 44 of them are affected with thyroid disease which usually progresses to hypothyroidism if left untreated.2,3,6-8 The prevalence of both diseases has increased rapidly over the last decade despite the collective efforts of conscientious breeders working with veterinarians to test and screen out carriers and affected stock from their breeding programs.6,7,9
GENETIC AND CLINICAL EXPRESSION
1. Inherited (Congenital) vWD
vWD is the most common mild inherited bleeding disorder of humans and animals.2,7 The disease is an autosomal trait with variable clinical and genetic expression.1 In most affected purebreds or cross-breds, vWD is classified as Type I and is an autosomal incompletely dominant trait, which means it has variable expression within affected families.5-7 The bleeding tendency can be manifested either by homozygotes that have inherited the gene from both parents, or by heterozygotes that received the gene from either parent. Homozygosity is rarely seen in type I vWD, however, because affected puppies usually die during fetal development or shortly after birth.1
There is also an autosomal recessive form of vWD (type III) in which clinically affected individuals are homozygous for the vWD gene and have two asymptomatic, heterozygous (carrier) parents. Dogs affected with type III vWD produce virtually no von Willebrand factor (vWF) protein. The recessive disease has been recognized in Poland-China swine, Scottish terriers, Chesapeake Bay retrievers, Shetland sheepdogs, Drahthaars (German wirehaired pointers), and several individuals of other breeds.1,2,6,10 In some cases, animals clinically affected with type III vWD are actually the double heterozygous offspring of parents genotypically heterozygous for type I vWD (e.g. Shetland sheepdogs).6
A rare type of vWD (type II) has been discovered in German shorthaired pointers and a family of quarterhorses.10,11 In this form of vWD, affected individuals produce an abnormal dysfunctional vWF protein.
The type I disease is much more common than the other types of vWD and has been recognized in more than 50 breeds to date (Table I).7 Breeds with a high prevalence of the vWD gene include the Doberman pinscher (80%), Scottish terrier, miniature poodle, Pembroke Welsh corgi, German shepherd, Rottweiler, standard and toy Manchester terrier, Keeshond, standard and miniature dachshund (15-45%).2 A severe form of vWD has been documented in a Himalayan and a Siamese cat.2,3
vWD affects many animals although relatively few have severe problems and even fewer die. Bleeding typically involves mucosal surfaces.5,7,10 The bleeding episodes are worsened by physical, emotional and physiological stresses as well as by other concomitant diseases. Typical clinical signs include: recurrent gastrointestinal hemorrhage with or without diarrhea; recurrent hematuria; epistaxis; bleeding from the gums, vagina or penis; lameness that mimics eosinophilic panosteitis; stillbirths or neonatal deaths (“fading pups”) with evidence of bleeding at necropsy; prolonged bleeding at estrus or after whelping; bruises or hematomas on the surface of the body, limbs, or head; excessive umbilical cord bleeding at birth; and excessive bleeding from toe nails cut too short, or after tail docking, ear cropping and dewclaw removal. Affected dogs may bleed to death from surgical procedures.
2. Acquired vWD
In dogs, vWD becomes more clinically severe if the dog also has hypothyroidism.2,4,6-8 Thus, healthy carriers of the vWD gene may exhibit a bleeding tendency if they subsequently become hypothyroid, a situation commonly seen in many of the affected breeds. The relatively high incidence of both vWD and hypothyroidism combined with research on the synthesis and metabolic regulation of thyroid hormones and vWF have confirmed this link. 3,4,6-8 Hypothyroid dogs may also have thrombocytopenia which can contribute to mucosal surface bleeding. The bleeding tendency and laboratory abnormalities associated with hypothyroidism and vWD usually, but not always, resolve after treatment with appropriate doses of L-thyroxine.2,3,8,13 It is generally impossible to distinguish between the inherited and acquired types of vWD in an individual patient with currently available techniques, 4,7 although it would be highly unlikely for a dog affected with acquired vWD to have zero or undetectable levels of vWF:Ag. 7,8
In Doberman pinschers, there is a very high prevalence of vWD along with an increased frequency and severity of bleeding episodes when thyroid disease is present concurrently. 2,5-8,13,14 About 80% of the more than 20,000 Dobermans tested have < 50% von Willebrand factor antigen (vWF:Ag) and 18% of this total group exhibits a bleeding tendency.2 Daily thyroid supplementation can lessen or control the clinical signs in dogs with both diseases, and in some cases increases vWF:Ag levels 2- or 3-fold.2,4,8,13 Furthermore, circulating antithyroid antibodies are often present in humans and dogs with autoimmune thyroiditis (Hashimoto’s disease) several years before the lymphocytic thyroid disease becomes clinically apparent. Animals with thyroid dysfunction can therefore have fluctuating levels of vWF and, when placed on thyroid supplementation, levels can increase to within normal limits. This could preclude the accurate diagnosis of their genetic status for vWD (i.e., carriers of vWD might test as normal when on thyroid medication). Because of the exacerbation of bleeding tendencies in dogs with vWD and hypothyroidism, breeders and veterinarians need to be aware of the increased risk and be more cautious about breeding or performing surgery on dogs with both problems. 7,8
The accurate diagnosis of vWD requires specialized vWF assays because the routine coagulation screening tests (APPT, PT and thrombin time) are nondiagnostic.2 Affected individuals have long bleeding times, and definitive diagnosis is made by finding reduced or undetectable plasma concentrations of vWF:Ag and abnormal platelet-related assays of vWF.5-7,9 Animals affected with the recessive type III disease are homozygotes having immeasurable vWF:Ag(0% vWF:Ag) and both parents are heterozygotes having reduced levels vWF:Ag (< 50% of normal).1,2,7 By contrast, animals affected with the incompletely dominant type I disease are heterozygotes having reduced but measurable vWF:Ag (1-49%).7 Diagnosis of the rare type II form of vWD usually requires analysis of vWF multimer forms.10,11 Table 2 summarizes the diagnostic finding in vWD.
Assays of vWF protein are used clinically to diagnose individuals affected with vWD, and as a means of identifying asymptomatic carriers of the vWD trait. The vWF:Ag assay is a quantitative measure of the protein’s antigenic properties. This assay has been used routinely as a screening test for vWD.9
Until recently, most clinical laboratories used the Laurell electroimmunoassay methodology to measure vWF:Ag.15 Alternative quantitative methods based on radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques are now available.14-18 The ELISA methodologies provide parallel accuracy to the Laurell method but are more sensitive (measure below 1% vWF:Ag versus a lower detection limit of 7% by Laurell).17 Additional benefits of the ELISA include increased efficiency and ease of standardization.
Our Albany, NY laboratory switched over in 1990 to a newly developed ELISA system for measuring vWF:Ag.16,17 We compared the vWF:Ag level determined by ELISA and Laurell techniques of over 600 randomly chosen, individual canine plasma samples submitted for testing.16 We also looked closely at subsets of normal and affected dogs. In most individuals the levels of vWF:Ag measured by ELISA was essentially the same as that determined by Laurell (correlation coefficient of 0.87). In a small number of cases (1-3%) the results of the two assays were discordant; that is higher or lower vWF:Ag values were obtained on the same sample when measured with both techniques. These differences most likely reflect slight antigenic variations in part of the vWF molecule in these dogs, a situation also recognized in humans.16 As more data are collected, including the results of pedigree analysis, vWF protein characterization and thyroid testing, we hope to better understand the significance of these few discordant results.
A group of commercial veterinary testing laboratories now offer vWD testing. Most of them send their samples directly to our Albany, NY laboratory but a few do their own testing. Some human clinical laboratories also offer veterinary testing. It is advisable to be cautious here because routinely used human assays need to be adapted for use in animals to give meaningful results. A laboratory performing the test in-house should validate the assay for measuring canine vWF, check sample type and quality and note any unusual findings on the report. It is important to strictly standardize quality control and review procedures to minimize chances for an occasional error. The reproducibility of the ELISA vWF:Ag assay at our laboratory is + 8% (intra-and interassay variation) with a coefficient of variation of less that 9%.15,17
Influence of Sample Collection, Processing and Transport
Errors or difficulties encountered when collecting and processing blood samples for vWD screening still remain the most common reason for inconsistent results. Samples containing significant hemolysis or clots, even small ones, indicate that the animal was stressed and/or the sample was not obtained cleanly. Results for vWF:Ag on these samples may be artificially raised, lowered, or invalid.15 One way to assure more consistent interpretation of vWD genotype for a breeding program is to retest all foundation stock if there is or was any question about the sample quality on an original test. Having more than one test result provides more confidence about the true reading of valuable foundation breeding stock.9
Progeny testing provides a means of verifying genotypic classification based on measurements of vWF:Ag. Results of vWF:Ag for both parents and entire litter(s) should be provided for accurate interpretation. The expected outcome from matings of two parents genetically normal for the vWD trait is an entire litter of normal puppies. The presence of one or more carriers in such matings indicates misclassification of one or both parents.
When transporting samples for testing they should stay cold or frozen in transit; an overnight mail or package express system is required. Samples should be placed in an insulated container with a frozen cold pack even in wintertime, because during transport the box could be kept indoors near a heating unit.15
Disparity Between Duplicate or Repeat Test Results
The most frequent cause of divergent results between so-called “duplicates” is that the specimens are not truly duplicates. Common mistakes in sending duplicates include taking separate samples from different veins one after the other (because the animal is more stressed for the second one), or dividing the plasma after centrifugation by removing the top portion and putting it in one tube and placing the bottom portion in a second tube. As plasma proteins centrifuge according to weight and mass, the heavier plasma proteins are at the bottom and lighter ones at the top. The correct was is to remove all the plasma, mix it thoroughly and then divide it into two portions.
Another common problem arises when VacutainersR are used to collect the blood sample. These tubes are designed for drawing the blood directly through the needle by vacuum into the tube. Many veterinary clinics, however, collect blood first into a dry, empty syringe and then add it to the tube containing the citrate anticoagulant. This modification allows for clotting to begin before anticoagulation occurs, and so samples are activated or partially clotted which can produce inaccurate vWF:Ag readings.15
Production of vWF and Relationship to Autoimmune Thyroid Disease
The endothelial cells lining blood vessels are the primary and essentially sole source of vWF production in the dog, and are the major source in other species.3,7,8,10 Any disease process that alters endothelial metabolism and protein synthesis can affect production and/or secretion of vWF.8,13 Autoimmune diseases, especially thyroid disease, can disrupt vWF production and function resulting in the secondary, acquired for of vWD.4,8,13 Therefore, lower levels of vWF are frequently, but not always, seen in dogs affected with or developing thyroid disease.
Blood Testing for vWD
The accuracy of the testing program in detecting the vWD genotype has been evaluated by retrospective analysis of the results to date for all three mating types (normal X normal; normal X carrier; and carrier X carrier).9 The rate of misclassification of genetic status by this test was found to be less that 5%, which means that accurately measured levels of vWF:Ag are a reliable predictor of vWD genotype in healthy animals.6,9 However, misclassification of genotype either way (false positive or false negative) is always possible. Some of the reasons for misclassification are:
1) the animal belongs to the 5% not identifiable by this test;
2) some other health problem such as hypothyroidism has altered vWF:Ag levels or the animal is receiving thyroid supplementation;
3) the animal was in heat, pregnant or lactating when tested;
4) one or both parents have been incorrectly identified as being the true parent or as having a normal vWD genotype; or
5) a collection, processing, or laboratory error was made. This last reason accounts for most of the misclassifications involving any type of genetic screening program.9
If a breeder plans a mating with another person’s stud dog or brood bitch, wishes to lease or buy a tested animal, or to buy a puppy of vWD tested parents, it is prudent to advise them to see a copy of the results of vWF testing indicating the dog(s) test status beforehand, rather than rely on the memory or word of the owner. This is an added precaution and should not imply dishonesty or mistrust.
Test Results and Interpretation
Results of our Comparative Hematology Laboratory vWD testing program are reported as the percentage of vWF:Ag in comparison to an established pooled plasma standard from normal healthy animals of the species being tested. The ranges for measurement of vWF:Ag utilizing the ELISA technique are basically the same as those previously established for animals with the Laurell technique (Table 2).15-17
Dogs testing in the normal range are at low risk of transmitting the vWD trait. Dogs that are free of the vWD trait, when bred to a normal testing mate, should only produce offspring of normal genotype (i.e. with vWF:Ag levels > 79%).
When using the vWF:Ag test as a genetic predictor of vWD status, certain restriction need to be applied before one considers test results falling within the normal range as valid. Measurements of vWF:Ag performed on animals that are ill or vaccinated within 10-14 days, or on females during heat cycle or pregnancy and nursing may be transiently higher or lower than the true baseline, and therefore, may cause misclassification. Animals with endocrine disease, especially thyroid dysfunction can also have fluctuating levels of vWF. 6-8,13
There is an overlap region of vWF:Ag test levels (borderline range) between normal animals and those testing as carriers of vWD. Animals that test in this range cannot be accurately classified as free of the vWD trait or carriers on the basis of that measurement. We recommend that those testing in this range be bred only to mates testing well within the normal range. Their offspring should be tested to identify normal individuals and to further clarify the parent’s vWD status. Healthy dogs testing in the abnormal range are considered to be carriers of the vWD trait and can transmit the defect to some of their offspring.5,9
We have seen a dramatic increase since 1985 in the number of purebred dogs having low levels of vWF:Ag (<50%). When the data for Scottish terriers and Shetland sheepdogs were analyzed, a statistically significant increase in the prevalence of the vWD trait was found representing both inherited and acquired deficiency of vWF.7 This overall increase is real because the number of dogs tested each year has continued to be large enough for statistical validation.7 Several explanations could account for this increase. The first invokes a shift in the measurement of vWF:Ag; this possibility is ruled out because our assay has been standardized over time and so varies less than + 8% of the actual value. Secondly, the prevalence of inherited vWD is increasing because tested or untested carriers continue to be bred as the popularity of these breeds is maintained. Lastly, the increasing prevalence of thyroid disease has produced a parallel increase in acquired vWD. The most likely explanation is a combination of the last two possibilities.7
MANAGEMENT AND TREATMENT
Proper management and treatment of bleeding disorders cannot be achieved without an appropriate physiologic and physical environment for hemostasis, tissue repair, and prevention of recurrence. An extremely important aspect of medical management is to avoid the use of drugs know to interfere with hemostasis (e.g. aspirin, phenylbutazone, potentiated sulfonamides, estrogens, promazine derivatives, penicillin G).1,2,4 These drugs are contraindicated for patients with moderate or severe hemostatic defects since they impair platelet function and further compromise the stability of the hemostatic plug.2
Live-virus vaccines and viral infections also affect platelet and/or endothelial function.2-4 The effect occurs during the viremic phase after vaccination or exposure (usually at 5-10 days) and results in a relative thrombocytopenia, which may prolong the bleeding time and predispose the animal to hemorrhagic problems. Platelet reductions of 100,00/ul can occur. During this period animals with hemostatic defects are at risk and should be watched for signs of bleeding. Elective surgical procedures such as ear cropping, spaying, castration, and dental surgery should be performed within 48 hours after vaccination or postponed for 10-14 days.2
Until recently, the limited availability of blood products for routine use in clinical veterinary medicine has necessitated the development of alternate strategies for the treatment and management of bleeding disorders.13,18 Table 3 outlines a stepwise approach for treatment of vWD. In those breeds in which both vWD and hypothyroidism occur relatively often, the administration of oral thyroid hormone (e.g. 0.1 mg/10 lbs. body weight given twice daily) has been effective in controlling bleeding episodes exacerbated by these concurrent diseases.2,4,6-8,13 Clinical signs of bleeding are often lessened or controlled within 48 hours after therapy is initiated.8,13 The therapeutic response should be always monitored with serial toenail or other bleeding times and periodically by thyroid function tests. Dosages of thyroid medication should be adjusted accordingly to maintain thyroid levels of total T4, free T4 and total T3 within the upper half to third of the adult normal ranges.4,13 In humans, L-thyroxine therapy has been shown to reduce levels of the circulating antibodies responsible for thyroiditis and hypothyroidism.13 Presumably this plays a role in the observed reversal of the bleeding tendency associated with thyroid disease. In dogs, the bleeding time is dramatically shortened and in come cases may even be corrected to within normal limits (<5 mins) from pre-treatment values beyond 20 mins.13 Thus, thyroid supplementation alone may suffice to control bleeding in mild to moderate vWD, a situation analogous to the use of desmopression (DDAVP)or danazol to control bleeding in humans.4,10,13,14 DDAVP treatment has recently been shown to improve vWF levels in normal dogs and Doberman pinschers with vWD,14,18 although relatively high doses are required and the response is transient (1-3 hours).4,13,18 Although most Doberman pinschers benefit from DDAVP treatment, certain individuals fail to respond, and the effect of this drug in other affected breeds has not been determined.18 Hormonal therapy with DDAVP or thyroxine cannot induce vWF production in homozygous, congenitally vWF-deficient dogs having type III vWD (zero level of vWF:Ag) and may not be beneficial in severe type II vWD.13,18
Transfusion of fresh-frozen plasma or plasma cryoprecipitate along with packed red cells, where indicated, remains the treatment of choice for control of severe bleeding crises in vWD, for homozygous affected vWD patients undergoing surgery, and for dogs that fail to respond adequately to L-thyroxine or DDAVP therapy.2,13,18
1. Dodds, WJ. von Willebrand’s disease in dogs, Mod Vet Pract 65: 681-686, 1984.
2. Dodds, WJ. Bleeding disorders. In: Handbook of Small Animal Practice. RV Morgan (ed.), Churchill Livingstone Inc., New York, NY, 1988, pp. 773-786; 2nd ed., 1992, pp. 765-777.
3. Dodds, WJ. Contributions and future directions of hemostasis research. J Am Vet Med Assoc 193: 1157-1160, 1988.
4. Dodds, WJ. Bleeding and immune diseases, Parts I and II, and acquired von Willebrand’s disease. Proc. 56th meeting AAHA, St. Louis, MO, 1989, pp. 606-619.
5. Brooks, M, Dodds, WJ, Raymond, SL, Catalfamo, J. von Willebrand’s disease: signs, causes, treatment. Dogs in Canada, October 1989, pp. 74-77.
6. Raymond, SL, Jones, DW, Brooks, MB, Dodds, WJ. Clinical and laborartory features of a severe form of von Willebrand disease in Shetland sheepdogs. J Am Vet Med Assoc 197: 1342-1346, 1990.
7. Brooks, M, Dodds, WJ, Raymond, SL. Epidemiologic features of von Willebrand’s disease in Doberman pinschers, Scottish terriers and Shetland sheepdogs, 260 cases (1984-1988). J Am Vet Med Assoc 200: 1123 1127, 1992.
8. Avgeris, S. Lothrop, CD Jr, McDonald, TP, Plasma von Willebrand factor concentration and thyroid function in dogs. J Am Vet Med Assoc 196:921-924, 1990.
9. Dodds, WJ, Moynihan, AC, Fisher, TM, and Trauner, DB. The frequencies of inherited blood and eye diseases as determined by genetic screening programs. J Am anim Hosp Asso 17: 697-704, 1981.
10. Johnson, GS, Turrentine, MA, Kraus, KH. Canine von Willebrand’s disease: a heterogenous group of bleeding disorders. Vet Clin North Am 18:195-229, 1988.
11. Brooks, M, Leith, GS, Allen, AK, Woods, PR, Benson, RE, Dodds, WJ. A bleeding disorder (von Willebrand’s disease) in a quarterhorse. J. Am Vet Med Assoc 198: 114-116, 1991.
12. French, TW, Fox, LW, Randolph, JF, Dodds, WJ. A bleeding disorder (von Willebrand’s disease) in a Himalayan cat. J Am Med Assoc 190: 437-439, 1987.
13. Dodds, WJ. Blood substitutes. In Cotter, SM. Comparative Transfusion Medicine. Adv Vet Sci Comp Med 36: 257-290, 1991.
14. Meyers, KM, Wardrop, KF, Dodds, WJ, Brassard, J. Effect of exercise, DDAVP and epinephrine on factor VIII:C/von Willebrand factor deficient Doberman pinscher dogs. Thromb Res 57:97-108, 1990.
15. Benson, RE, Jones, DW, Dodds, WJ. Efficiency and precision of electroimmunoassay for canine factor VIII-related antigen. Am J Vet Res 44:399-403, 1983.
16. Benson, RE, Catalfamo, JL, Brooks, M, Dodds, WJ. A sensitive immunoassay for von Willebrand factor. J Immunoassay 12: 371-390, 1991.
17. Benson, RE, Catalfamo, JL, Dodds, WJ. A multispecies enzyme-linked immunosorbent assay for von Willebrand factor. J Lab Clin Med 119: 420-427, 1992.
18. Kraus, KH, Johnson, GS. von Willebrand’s disease in dogs. In: Current Veterinary Therapy X, Small Animal Practice. RW Kirk (ed.), WB Saunders Co., Philadelphia, 1989, pp. 446-451.
BREEDS OF DOGS KNOWN TO BE AFFECTED WITH VON WILLEBRAND’S DISEASE (as of March 1992)
A. Breeds with a High Prevalence of vWD Gene (15-80%)
B. Breeds with Either a Lower or Unknown * Prevalence of vWD Gene
Afgan (sic) Hound Great Pyrenees
Airedale terrier Greyhound
Akita Irish setter
Alaskan Malemute Irish woldhound
American cocker spaniel Italian Greyhound
Australian cattle dog Kuvasz
Azawakh Labrador retriever
Bearded collie Lakeland terrier
Bichon frise Lhasa apso
Boxer Old English sheepdog
Cairn terrier Samoyed
Chesapeake Bay retriever Shih tzu
Collie Siberian husky
Drahthaar (German Skye terrier
wirehaired pointer) Soft-coated Wheaten terrier
English cocker spaniel Swiss mountain dog
English setter Tibetan terrier
English springer spaniel Vizsla
Fox terrier-smooth Yorkshire terrier
Fox terrier-wire Whippet
German shorthaired pointer Great Dane
* Too few studied to date to estimate prevalence.
DIAGNOSIS OF VON WILLEBRAND’S DISEASE*
von Willebrand Factor
Antigen (%) +
70 or greater
Normal range is 70-180%
Lower end of normal range; caution advised for breeding stock. Mates should have higher levels and pups should be checked.
Borderline normal (equivocal result) or heterozygous carrier of vWD gene. Recommend retesting and/or breeding only to higher testing mates. Pups should be checked
Less than 50
Type I vWD. This is the most common form of vWD. Heterozygous carrier of vWD.* Recommend breeding only to higher testing mates. Pups should be checked.
Less than 50
Type I vWD. Has exhibited some bleeding problem (e.g. hematuria, epistaxis, melena, post-surgical bleeding).* Clinically afected animals should not be used for breeding.
N or Elevated
Less than 50
Type II vWD. Clinically affected animals exhibit severe bleeding symptoms. Multimeric analysis of vWF reveals absence of high molecular weight multimers. These animals should not be used for breeding. Has been report in German shorthair pointers.
Less than .01
Type III vWD. Exhibits bleeding symptoms and is homozygous for vWD. This animal is the product of two asymptomatic, heterozygous carrier parrents and should not be used for breeding.
Greater than 180
Probably reflects stress, an improper sample, or activation from disease. Recommend retesting. Test invalid for prediction of genetic status for vWD trait.
+ Formerly called factor VIII-related antigen; measured by ELISA methodology. When measured by Laurell or FIA methods, normal ranges may vary slightly.
* Development of concomitant thyroid dysfunction may aggravate exisiting vWD or increase the risk of bleeding in some previously asymptomatic carriers.
** Abnormal bleeding time is not specific for vWD; hemostatic disorders including thrombocytopenia and platelet dysfunction will also cause variable prolongation of bleeding time tests.
TREATMENT OF VON WILLEBRAND’S DISEASE
For short-tem control of bleeding or prophylaxis for dogs at risk to bleed from vWD:
1. For elective surgery or invasive procedures, assess bleeding potential first with a toenail or buccal mucosal bleeding time. Normal values for dogs are 2-5 minutes.
2. UseL-thyroxine (T4) therapy at 0.1 mg/4.5 kg body weight twice daily for 7-10 days, if the bleeding time is prolonged or the patient is bleeding. Start 48 hours prior to elective surgery where applicable. Recheck bleeding time before performing procedure to ensure adequate hemostasis. Continue thyroid replacement beyond 10 days if patient is still bleeding or has thyroid disease. Thyroid supplementation is believed to promote hemostasis by improving platelet function, stimulating thrombopoiesis in bone marrow and other sites, and enhancing protein synthesis/release of vWF and other coagulation factors.13
3. An alternative drug for elective procedures is DDAVP (desmopressin) which causes a transient correction of the bleeding time in most dogs with vWD. Recommended dosage is 1 ug/kg given subcutaneously 20 minutes before anesthesia.18
4. Transfuse fresh-frozen plasma 6-10 ml/kg of body weight once or twice daily, if bleeding continues or bleeding time is still prolonged after use of L-thyroxine. When available, cryoprecipitate* (cryoconcentrated plasma) can be used at doses equivalent to double those of whole plasma [12-20 ml/kg body weight] because an average 50% loss of activity occurs during preparation. For elective procedures (e.g. surgery on Doberman pinschers with vWD), transfuse first and then perform surgery within 4 hours because this period provides the maximum correction of the bleeding time.
5. For patients with PCV at or below 15%, transfuse packed red blood cells in saline at 6-10 ml/kg given once or twice daily. The packed cells can also be mixed with fresh-frozen plasma and transfused as reconstituted whole blood.
6. Avoid drugs or biologics that can impair hemostasis and/or induce thrombocytopenia. These include:
trimethoprim-sulfonamides modified live virus vaccines (3-10 days afterwards)
promazine tranquilizers warfarin
* For control of severe bleeding crises or for homozygous affected vWD patients undergoing surgery. In most cases, bleeding in animals with vWD can be controlled or prevented by using L-thyroxine, DDAVP and/or fresh frozen plasma.
Original Doc: vwd04.doc
Mutation Causing von Willebrand's Disease In Scottish Terriers
Patrick J. Venta, Jianping Li, Vilma Yuzbasiyan-Gurkan, George J. Brewer, and William D. Schall
Source: Journal of Veterinary Internal Medicine 2000; 14:10-19. Submitted by Dr. Patrick Venta.
Von Willebrand's Disease (vWD) in the Scottish Terrier is a serious, often fatal, hereditary bleeding disorder. Elimination of the mutated gene by selective breeding is an important goal for the health of this breed. Although the standard protein-based tests are accurate for identification of affected Scottish Terriers, they are not reliable for the identification of carriers of the mutant gene unless multiple replicate assays are performed. A simple, highly accurate test for carriers of the disease is needed so that veterinarians can counsel clients on which animals to use in their breeding programs. The complete coding region of von Willebrand factor (vWF) complementary DNA (cDNA) was sequenced from an affected animal, and a single base deletion in the codon for amino acid 85 of the prepro-vWF cDNA that leads to Scottish Terrier vWD was identified. A highly accurate polymerase chain reaction assay was developed that can distinguish homozygous normal animals from those that are homozygous affected or het erozygous. In a voluntary survey of 87 animals provided by Scottish Terrier owners, 15 were carriers and 4 were affected with vWD, 2 of which had previously been shown to have undetectable vWF. The determination of the complete canine vWF CDNA sequence should facilitate the identification of additional vWD alleles in other breeds and other species.
on Willebrand's disease (vWD) is a bleeding disorder of variable severity that results from a quantitative or qualitative defect in von.Willebrand factor (vWF).1-5 vWD has been observed in many mammalian species, including humans and dogs. This clotting factor has 2 known functions: (1) stabilization of factor VIII (anti-hemophilic factor A) in the blood and (2) aiding the adhesion of platelets to the subendothelium, which allows them to provide hemostasis more effectively. An affected individual may bleed severely if the factor is missing or defective.
The disease is the most common hereditary bleeding disorder in both dogs and humans, and it is genetically and clinically heterogeneous. Three clinical types, 1, 2, and 3 (formerly I, II, and III6, 7), have been described. Type I vWD is thought to be inherited in a dominant, incompletely penetrant fashion in humans. In dogs, Type I vWD appears to be inherited in a recessive fashion in at least some breeds, although it is possibly inherited as dominant with incomplete penetrance in others. Bleeding appears to be due to the reduced amount of vWF rather than a qualitative difference. ___________________________________________
From the Departments of Small Animal Clinical Sciences (Venta, Yuzbasiyan-Gurkan, Schall) and Microbiology (Venta, Yuzbasiyan- Gurkan), College of Veterinary Medicine, Michigan State University, East Lansing, Ml and the Department of Human Genetics University of Michigan, Ann Arbor, MI (Li, Brewer). Preliminary findings re ported at the International Society for Animal Genetics meeting, 1996.
Submitted September 8, 1998; Revived May 6, 1999; Accepted Au gust 12, 1999.
Copyright (D 2000 by the American College of Veterinary Internal Medicine
Although type I is the most common form of vWD found in most mammals and can cause serious bleeding problems, it is generally less severe than the other 2 types.
Type 2 vWD patients have a qualitative defect in vWF, lacking high-molecular-weight multimers. Affected humans and dogs have a severe bleeding tendency.2, 5 Type 2 vWD is inherited in a dominant fashion in humans but is inherited recessively in dogs .4, 8 Type 3 VVID is the severest form of the disease. It is inherited as an autosomal recessive trait, and affected individuals have no detectable vWF in their blood. Serious bleeding episodes require transfusions of blood or cryoprecipitate to supply the missing vWF. Heterozygous carriers have moderately reduced factor concentrations but generally appear to have normal hemostasis.
Scottish Terriers appear to have only type 3 vWD.3, 4 Homozygotes have no detectable vWF and have a severe bleeding disorder. Heterozygotes have reduced amounts of the factor and are clinically normal.9 The prevalence of vVD among Scottish Terriers including both heterozygotes and homozygotes has been variously estimated from 18% to 30%.9-11
Currently, detection of affected and carrier Scottish Terrier dogs is done by vWF antigen testing12, 13 or historically by coagulation assays.14, 15 These procedures yield variable results because the protein-based tests can be influenced by such things as sample handling, estrus, pregnancy, vaccination, age, and perhaps hypothyroidism.16-21 A dog that tests within the normal range on 1 day can test within the carrier range on another day. This variability makes it difficult for breeders to use this information to eliminate the disease-causing allele from their lines. Thus, it is highly desirable that a more definitive test be developed for Scottish Terriers.
Herein we report (1) the complete amino acid sequence as inferred from the canine vWF CDNA, (2) the discovery of the mutation that causes vWD in Scottish Terriers, and (3) the development of a simple direct DNA test for this mutation that distinguishes homozygous normal ("clear"), carrier, and affected animals with complete accuracy. This test gives breeders the potential to dramatically reduce the frequency of the disease-causing allele and the incidence of the disease within the Scottish Terrier breed. In addition, the determination of the complete nucleotide sequence of the canine vWF CDNA should lead to the rapid development of molecular tests for other breeds and for other species.
Materials and Methods
Isolation of RNA
The source of the RNA was a uterus surgically removed because of pyometra from a Scottish Terrier affected with vWD (factor value < 0.1% and a clinical bleeder). Total RNA was extracted from the tissues using Trizol.a The integrity of the RNA was assessed by agarose gel electrophoresis on a 1 x TBE (1 x TBE: 90 mM Tris, pH 8.3, 90 mM borate, 1 mM EDTA), 1% agarose gel and inspection of intact 18S and 28S ribosomal RNA bands. The factor values for the animals reported herein (always less than the sensitivity of the assay for those affected) were determined by the Diagnostic Laboratory at the College of Veterinary Medicine of Cornell University, by the Comparative Laboratory of Hematology at the State of New York Department of Public Health, or by Drs. Read and Brinkhaus and their colleagues at the University of North Carolina and were reported by the owners.
Design of Polymerase Chain Reaction Pyimer Sets
Polymerase chain reaction (PCR) primers were designed for use in 2 regions of the gene where sequences from 2 species were available.22-23 These primers were designed using rules for cross-species amplifications.24 Most of the primers had to be designed for other regions of the gene using the human sequence alone.25 Primers were designed by taking into account number of codons per amino acid, relative amino acid mutabilities, and where applicable, conservation of nucleotide sequence. We used the same methodology to design a series of universal mammalian sequence tagged sites.24 Roughly 66% of the primers designed for the human sequence alone amplified the correct canine target. New primers had to be designed for regions that did not amplify with the original primer sets. After portions of the canine sequence had been determined, new primers were designed for the canine sequence. Primer sets and amplification conditions are available from the authors on request. Appropriate amplification conditions were determined using human and canine genomic DNAs for the initial primer sets. Generally, 94ºC for 1minutes, 57ºC for 2 minutes, and 72ºC for 3 minutes for 35 cycles worked well with the majority of the amplifications. All amplifications used Tris-HCl pH 8.3 (20ºC), 50 mM KCl, 1.5 mM MgCl2, 200 µM dNTPs, and 20 µLM primers in 25- or 100-µl reactions.
Reverse Transcyiplase PCR
Total RNA was reverse transcribed using random primers.16 Reverse transcription was performed using a commercially available kitb under the manufacturer's suggested conditions. Five microliters of reverse transcribed total RNA was included in each 50-µl amplification reaction. The cDNA was amplified using the primer sets shown to work on canine genomic DNA.
DNA Sequence Analysis
Amplification products were isolated from agarose gels by adsorption onto silica gel particles using the manufacturer's method.c Sequences were determined using 32P 5' end-labeled primers (the same ones used for the amplification) and a cycle sequencing kit,c The sequences of the 5' and 3' untranslated regions were determined after amplification using a RACE (random amplification of cDNA ends) kit.e Sequences were aligned using the Eugene software analysis package.f The sequence of canine intron 4 was determined from PCR- amplified genomic DNA. A "long PCR" kit was used to amplify this 5-kb intron.1
Design of a Diagnostic Test
PCR mutagenesis was used to create diagnostic and control BsiE * and Sau96 I restriction enzyme sites for the test. Amplification conditions for the test are 94°C for 1 minute, 61°C for 1 minute, and 72°C for 1 minute for 50 cycles using cheek swab DNA27 (Yuzbasiyan-Gurkan et al, unpublished). Twenty-five microliter reactions were run, and 1 µl of restriction enzyme was added directly to the PCR reaction and incubated for 3 hours at 60°C for BsiE (and 37°C for Sau96 I. Eight microliters of each reaction were loaded onto a 3:1 NuSieve: low EEO agarose 1 x TBE gel and run at 125 V for 1 hour. Gels were stained with ethidium bromide and photographed under 360-nM ultraviolet light.
A voluntary survey of Scottish Terriers was conducted to determine an estimate of the frequency of the mutation in the population. Participants were identified and enlisted through the efforts of members of the Scottish Terrier Club of America. All submitted samples was included in the survey. Previously determined factor values were not required, although some participants supplied values for their animals. DNA was collected from 87 Scottish Terriers from 16 pedigrees. DNA was isolated from blood using standard procedures26 or from cheek swab samples27 (Yuzbasiyan-Gurkan et al, unpublished). The genetic status of each animal in the survey was determined using the BsiE I test.
Comparison of the Canine and Human Sequences
The cDNA sequence has been placed in GenBank (accession no. AF099154). Two other groups have independently determined the canine vWF cDNA sequence and have also placed their sequences in GenBank (accession nos. L76227 and U66246). The alignment of the canine and human amino acid sequences is shown in Figure 1. There is 85.1% sequence identity between the prepro-vWF sequences of the canine and human amino acid sequences (Fig 1). The propeptide region is slightly less conserved than the mature protein (81.4% versus 87.5%). There were no other noteworthy sequence identity differences in other regions of the gene or between the known repeats contained within the gene (data not shown).
Fourteen potential N-linked glycosylation sites are present in the canine sequence, all of which correspond to similar sites contained within the human sequence. The 2 integrin-binding sites identified in the human vWF protein sequence 29 also are conserved in the canine sequence (Fig 1). The 3' untranslated region has diverged to a greater extent than the coding region (data not shown), a divergence comparable to that found between the human and bovine sequences derived for the 5' flanking region.30, 31
Two other groups independently determined the sequence for most of exon 28.32, .33 All 3 sequences are in complete agreement, although we have also found 2 silent variants in other breeds (unpublished data). Partial sequences of exons 40 and 41 (cDNA nucleo-tides 6923-7155, from the initiation codon) were also
independently determined by another group as part of the development of a polymorphic simple tandem repeat genetic marker.34 There is a single nucleotide sequence difference between this sequence (T) and ours (C) at nucleotide position 6928.
Scottish Terrier vWD Mutation
A single base deletion was found in exon 4 (Fig 2). This frameshift mutation at codon 88 leads to a new stop codon 103 bases downstream. The resulting severely truncated protein of 119 amino acids does not include any of the mature von Willebrand factor region. The identity of the base in the normal allele was determined from an unaffected dog.
Human MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYL 60
Dog -S-T-LVR ----------K--TK--V ---M-----L-G--I ---E-------D----
Human LAGGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYL 120
Human ETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTL 180
Dog -A -------S-----------N-----------------------------K-------
Human TSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPL 240
Dog ------------------R-K-V ----P--V--D-V-QV ---------A---------
Human VDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGME 300
Human YRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDZGLCVESTECPCVHSGKRYPPG 360
Human TSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQD 420
Human HSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDVQLPLLKGDL 480
Human RIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSG 540
Human LAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVS 600
Human PLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQ 660
Human CGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPED 720
Human IFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADN 780
Human LRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPCFHQGKEYAPGE 840
Human TVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGS 900
Human NPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGR 960
Human YIILLLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVD 1020
Human FGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPY 1080
Human LDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGY 1140
Human ECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCE 1200
Fig 1. Comparison of the human and canine prepropeptice vWF amino acid sequences. The asterick indicates the location of the Scottish Terrier vWD mutation. Potential N-linked glycosylation sites are shown in bold. The known and postulated integrin-
Human VAGRRFASGKKVTLNPSDPEHCQICRCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVE 1260
Human DISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVE 1320
Human YHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRI 1380
Human ALLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVL 1440
Human SSVDELEQQRDEIVSYLCDLAPEAPPPTLPPHMAQVTVGPGLLGVSTLGPKRNSMVLDVA 1500
Human FVLFGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGD 1560
Human ILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLP 1620
Human GDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTL 1680
Human SPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITT 1740
Human IDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILV 1800
Human TDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTM 1860
Human VTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLXTHRVNCD 1920
Human RGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNK 1980
Human EQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEV 2040
Human NVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRD 2100
Human GTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFY 2160
Human AICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHC 2220 Dog -M--P----PKK---A--L-------K-------PAN---------------------L-
Human DGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQI 2280
Human CTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHC 2340
Fig 1. (continued) binding sites are boxed. Amino acid numbers based on the prepropeptide vWF sequence are shown on the right side of the figure. The mature protein begins at position 763. The human sequence is derived from GenBank accession no. X04385.46The canine nucleotide sequence has been deposited in GenBank (accession no. AF099154).
Human ERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCOEYECACNCVN 2400
Human STVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAV 2460
Human MGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGSQ 2520
Human WASPENPCLINECVRVKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSACCPSCRCERME 2580
Human ACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGEC 2640
Human CGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHK 2700
Human CLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMY 2760
Human SIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK 2813
Fig 1. Continued.
Development of a Diagnostic Test
A PCR primer was designed to produce a BsiE I site in the mutant allele but not in the normal allele (Fig 3). To ensure that the restriction enzyme cut the amplified DNA to completion, an internal control restriction site common to both alleles was designed into the nondiagnostic primer. The test was verified by digestion of the DNA from animals that were affected, obligate carriers, or normal (based on reports of factor values >100% of normal from commonly used testing labs and reported to us by the owners and from breeds in which type 3 vWD has not been observed). The expected results were obtained (e.g., Fig 4). Five vWD-affected animals from a colony of mixed-breed dogs founded from Scottish Terriers35 were homozygous for this mutation. An additional unaffected animal from this same colony was clear.
It would still be possible to misinterpret the results of the test if restriction enzyme digestion were incomplete and if the rates of cleavage of the control and diagnostic sites were vastly different. The rates of cleavage of the 2 BsiE I sites were examined by partially digesting the PCR products and running them on capillary electrophoresis. The rates were nearly equal (the diagnostic site is cut 12% faster than the control site).
The mutagenesis primer was also designed to produce a Sau96 I site in the normal allele but not the mutant allele. This test is the reverse of the BsiE I-dependent test with respect to which allele is cut. Natural internal Sau96 I sites serve as digestion control sites (shown in Fig 3). The test using this enzyme produced genotypic results identical to those using BsiE I for all animals examined (data not shown).
Three pedigrees were examined in which the normal and mutant alletes were segregating. The alleles, as typed by both the BsiE I and Sau96 I tests, showed no inconsistencies with Mendelian inheritance. One of these pedigrees included 2 affected animals, 2 phenotypically normal siblings, and the obligate carrier parents. The 2 parents were found to be heterozygous by the test, the 2 affected animals were found to be homozygous for the mutant allele, and the normal siblings was found to be heterozygotes (Fig 4).
Population Surpey for the Mutation
Cheek swabs or blood samples were collected from 87 animals to determine the prevalence of carriers in the US Scottish Terrier population. Although we attempted to make the sample as random as possible, the results of the tests revealed that these dogs came from 16 pedigrees, several of which were distantly interconnected. This close connection is due to some aseertainment bias based on ownership (as opposed to phenotypic ascertainment bias). In these 87 animals, we found 4 affected and 15 carrier animals.
The determination of the canine vWF CDNA sequence permits the first comparison between 2 complete inferred mammalian vWF protein sequences. The protein has a structure based on 4 kinds of repeat units: the A, B, C, and D repeats.1, 2 There does not appear to be a strict correspondence between these repeat units and the intron-exon structure of the gene. The most notable feature of the gene is the large exon 28, which contains most of the 3 A repeat units. Although most human type 2 mutations occur in this exon, type 3 mutations seem to have a relatively random distribution over the entire length of the gene. Although we have not determined the entire intron-exon structure of the canine gene, the few intron splice sites that we have determined are suggest that the human and canine gene structures are very similar (unpublished results).
The order and length of these repeats has remained conserved between the 2 species. There are no inserted or deleted amino acids, and thus the lengths of the human and canine prepropeptide sequences are identical at 2,813 aniino acids. The integrin receptor sites of the human factor are both conserved in the canine molecule (Fig 1). All of the 234 cysteine residues are conserved between the 2 species, suggesting that these residues are of importance for the maintenance of the structure of the molecule. The determination of the canine sequence will also allow cross-species PCR amplifications to be conducted more easily, which should lead to a greater understanding of the structure, function, and evolution of this important coagulation protein.
The results reported here also establish that the single base deletion found in exon 4 of the vWF gene causes vWD in the Scottish Terrier breed. The putative protein produced from the mutant allele is extremely short and does not include any of the mature vWF protein. Four Scottish Terriers known to be affected with the disease were homozygous for the mutation. Five other mixed-breed dogs descended from Scottish Terriers and affected with vWD were also homozygous for the mutation. No phenotypically normal animals were homozygous for the mutation. Unaffected obligate carriers are always heterozygous for the mutation.
The gene frequency in the population surveyed appears to be around 0.13, resulting in a heterozygote frequency of about 23% and expected frequency of affected animals of about 2%. It is extremely difficult to obtain truly random samples from domestic animal populations, and thus caution is needed in extrapolating the results from the survey conducted in this report to the whole Scottish Terrier population. Although the sample size is relatively small and probably biased in unknown ways, these data are in general agreement with the results of protein-based surveys5, 10 and demonstrate that the mutant allele is not uncommon.
The data collected so far indicate that this mutation could account for most and possibly all of the VVVD found in Scottish Terriers. This result is consistent with the allelic homogenity found for other genetic diseases of comestic animals that have been defined at the molecular level.36-38 This homogenity is most likely due to the pronounced founder effect (usually popular sire effect) that occurs in domestic animals, in contrast to the situation in most human and wild animal populations.
Other vWD alleles may exist or may occasionally arise in the Scottish Terrier breed. However, seveal facts suggest that the mutation described in the current report accounts for most of the disease in the breed: (1) the number of carriers for this mutation is of the same magnitude as that found in previous protein-based surveys, 4, 9, 10 which would be consistent with the characterization of this mutation as the major mutation; (2) all of the results obtained in this study are explained by the 1 mutation; (3) only type 3 vWD has ever been reported in Scottish Terriers, which supports the probability of allelic homogeneity; and (4) the founder effect is known to be an important element in domestic animal genetic diseases. Although there is still a possibility that other vWD alleles exist at low frequency in the Scottish Terriers, it seems likely that the elimination of this 1 mutation would cause vWD to become a rare disease in the breed.
Published data using the protein-based factor assays have shown that, at least in several instances, obligate carriers have had factor values that would lead to a diagnosis of "clear" of the disease allele. For example, in 1 study an obligate carrier had a factor value of 78%.39 In another study, at least some of the obligate carriers had factor values of >65%.35 In both of these studies, however, researchers used older method-ologies. (Laurell electrophoresis and venom coagula-tion), although even enzyme-linked immunosorbent assays can produce results with considerable variation.40 In addition, the number of animals
Clear Carrier Affected
G A T C G A T C G A T C
Fig 2. Nucleotide sequencing ladders for the vWD mutation region for clear, carrier, and affected Scottish Terriers. The sequences were obtained directly from PCR products derived from genonmic DNA in exon 4. The arrowheads show the location of the C nucleotide that is deleted in the disease-causing allele. The carrier ladder of each base above the point of the mutation has a doublet appearance, as predicted for deletion mutations. The factor values reported by the owners of these Scottish Terriers were 54% clear, 34% carrier, and <0. 1% affected.
Fig 3. Molecular test to detect the Scottish Terrier vWD mutation. An asterick indicates the position of the deleted nucleotide. BsiE I restriction sites were created by PCR primer mutagenesis. The altered nucleotides in each primer are underlined. The normal and mutant allele can also be distinguished using Sau96 I. The naturally occurring Sau96 I sites are shown by double underlines. The highly conserved donor and acceptor dinucleotide splice sequences are shown in bold.
Fig 4. Scottish Terrier pedigree segregation of the mutant and normal vWF alleles. Exon 4 of the vWF gene was simplified from genomic DNA using PCR techniques. The PCR products were examined for the presence of the normal and mutant vWF alleles by agarose gel electrophoresis after digestion with BsiE I (see Fig 3). The affected animals are homozygous for the mutant allele (229 bp; lanes 3, 5). The other animals in this pedigree are heterozygotes (251 bp and 229 bp; lanes 1, 2, 4 , 6), including the obligate carrier parents. One hundred base-pair ladders were run in the marker lanes (M).
that fall into an equivocal range can be substantial. In 1 study, 19% of Scottish Terriers fell in this range (50-65% of the normal vWF antigen value).10 Thus, although the protein-based tests have been useful, the certainty of the DNA-based test should relieve the necessity of repeated testing and the variability associated with the protein-based assays. In a recent report, the authors provided evidence that daily biological variability is more important than the variability found in the test, and they stated that "multiple tests may be necessary to obtain a reliable estimate of vWF concentration in dogs."40
A direct test for the mutation should also be more useful than a linkage-based test using a vWF-specific polymorphic site, such as the known microsatellite marker contained within intron 40 of the canine vWF gene,34 because the mutation will probably be present on only a proportion of chromosomes that have a particular allele of the microsatellite. Although linkage disequilibrium between the mutation and 1 of the alleles may exist, there will still be many dogs with the same repeat number that do not have the mutation. In addition, a rare recombination between the mutation and the marker or a mutation in the microsatellite repeat could affect the accuracy of the diagnosis.
Although it should also be possible to deduce the genotypic status of the animal using family information, this method is less reliable than a direct test because it requires obtaining accurate information about relatives, which is often not possible. Genetic counseling using a linkage-based test requires considerably more effort, both on the part of the veterinarian or breeder and the service provider, than does analysis of blood or cheek swabs. Also, linkage-based tests do not consistently allow the determination of the genotype at the disease locus, often because the marker s not informative.41
The Scottish Terrier mutation is present in the propeptide portion of the vWF molecule. The propeptide is removed prior to delivery of the mature protein into the plasma. The propeptide is also important for the assembly of the multimeric mature vWF protein.42, 43 With the Scottish Terrier frameshift vWD mutation, neither the propeptide nor any of the mature factor would ever be produced. To our knowledge, no factor has ever been detected in the blood of affected Scottish Terriers, as predicted for the frameshift mutation that was identified.
The determination of the complete canine vWF cDNA sequence will have an impact upon the development of carrier tests for other breeds and other species. Currently, Shetland Sheepdogs and Dutch Kooikers are known to have a significant amout of type 3 vWD.9, 44 Type 3 vWD also has occassionally been seen in other breeds.39 All type 3 vWD mutations described in humans to date have been found within the vWF gene itself. The availability of the canine sequence will make it easier to find the mutations in other breeds. The mutation for the Dutch Kooiker breed has been recently reported.44 In addition, at leaset some type 1 mutations have been found within the human vWF gene, and thus type 1 mutations may be found within the canine vWF gene for breeds affected with that form of the disease. The availability of 2 divergent mallalian vWF cDNA sequences will also make it much easier to sequence the gene from other mammalian species using cross-species PCR methods.24, 45
The test that we have developed for the detection of the mutation in Scottish Terriers can be performed using only small amounts of DNA from any tissue. The tissues that can be obtained using the least invasive methods are blood and buccal cells. A cheek swab can be easily obtained by the veterinarian or breeder.
Care should be taken not to produce genetic bottlenecks when using results from this test. The production of affected animals can be avoided by using at least 1 clear animal in a mating. Genes conferring desirable characteristics in carrier animals can be maintained in a line until the disease-causing allele is lost after a few generations. Using this approach, genetic variability will be maintained at otherloci, and the potential detrimental effects of stringent selection against a deleterious gene will be avoided.
We thank Murat Gurkan, Paul Ferguson, Dr Yueying Cao, and Chris Willett for conducting some of the experiments tor this work and Dr Mary A. McLoughlin, Dr Stephen R DiBartola, Ms Helen Harbulak, Dr Majorie S. Read, Dr Kenneth M. Brinkhous, and Mrs Barbara DeSaye. This work was supported by the American Kennel Club, the Ortho-pedic Foundation for Animals, the Morris Animal Foun- dation, and the Companion Animal Fund at Michigan State University and by contributions from Scottish Terrier clubs and owners.
Some of the authors (GJB, PJV, VYG) have equity in the technology transfer company that they founded to pro vide the vV*9D test and other molecular genetic services to veterinarians and dog breeders.
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Original Doc: vwdgene.doc
von Willebrand's Disease in Dogs
The Doctor's Forum
Von Willebrand's disease is the most common, mild, inherited bleeding disorder of animals, and affects many breeds of dogs.
By W. JEAN DODDS, DVM
Division of Laboratories and Research, New York State Dept of Health, Empire State Plaza, Albany, NY 12201.
Adapted from the Veterinary Reference Laboratory Newsletter, Vol 7, No 3, 1983, courtesy of Veterinary Reference Laboratory, Inc.
The following material was submitted by a smooth fox terrier breeder who discovered VWD in her dogs. The article has been edited for readability and in intended for informational use only. For more in-depth information, use the References listed at the end of this article.
Source: The Fox Terrier Forum, April/May 1995, pp 4-7.
Von Willebrand's disease, the most common, mild, inherited bleeding disorder of animals, is a disease which generally has many physical characteristics but a low death rate. It affects many breeds of dogs. Clinical signs include hematuria, epistaxis, gingival or genital mucosal bleeding, lameness, and prolonged bleeding from cut nails or wounds. Hypothyroidism in a dog with von Willebrands will greatly increase the severity of the disease. Affected dogs and carriers should not be bred and dogs should be tested for von Willebrand's factor before breeding. Treatment of von Willebrand's involves intravenous infusion of fresh whole blood or plasma, at 3-5 ml/lb, with topical use of blood clotting compounds, and avoidance of drugs that interfere with blood clotting.
Von Willebrand's disease (VWD) is the most common, mild, inherited bleeding disorder of people and animals. It is a physical disease with two forms of clinical and genetic expression. It can be a disease that is physically recessive. In that event clinically affected individuals are homozygous for the VWD gene and have two asymptomatic, heterozygous (carrier) parents; or it can be a disease with an autosomal, incompletely dominant expression (variable penetrance), in which both homozygotes and heterozygotes can have a bleeding tendency. Homozygosity is often lethal in this form of VWD.
The recessive form of VWD has been recognized in Poland-China swine, Scottish Terriers and Chesapeake Bay Retrievers. The incompletely dominant for is much more common and has been recognized in 28 breeds of dogs, though virtually any breed may be affected. Several breeds have a high prevalence of the disease (15-60% gene frequency).
Many physical characteristics (but a low death rate) are generally associated with VWD. Typically, there is mild to severe bleeding, usually from mucosal surfaces. Bleeding is increased by physical, emotional and physiologic stress, hormonal imbalances, (especially hypothyroidism), and by other existing diseases in the effected dog such as parasitic, viral and bacterial infections. Typical clinical signs of VWD include: recurrent hematuria; epistaxis; gingival, vaginal or penile bleeding; lameness; stillbirths or neonatal deaths (“fading pups”), with evidence of bleeding at autopsy; prolonged estrual or postpartum bleeding; hematoma formation on the surface of the body, limbs or head; excessive umbilical cord bleeding at birth; and excessive bleeding from toe nails cut too short, or after tail docking, ear cropping or dewclaw removal. Severely affected dogs may bleed to death from surgical prodedures. A parvoviral infection often aggravates the bleeding tendency, which probably explains the high prevalence of severe parvoviral disease in Doberman Pinschers. About 58% of the Dobermans tested have the VWD trait.
RELATIONSHIP OF HYPOTHYROIDISM TO VWD
Several breeds of dogs have a high prevalence of hypothyroidism; the common occurrence of both VWD and hypothyroidism in some of these breeds (Dobermans, Golden Retrievers, Scotties, Corgis, Manchester Terriers) suggests a causal relationship between the synthesis and/or metabolic regulation of thyroid hormones and von Willebrand's factor (VWF) protein, which is deficient or abnormal in VWD. Hypothyroidism in people has been associated with a bleeding tendency caused by abnormal platelet function and low levels of factor VIII activity and VWF. Reduction in these abnormalities has been reported after oral thyroid supplementation. In our studies of Doberman Pinschers, there was an increased frequency and severity of bleeding episodes in animals with the VWD gene that were also hypothyroid. Clinical signs of bleeding in these dogs were lessened or controlled within 48 hours of initiating daily thyroid supplementation; VWF levels also increased. Thus, animals with thyroid dysfunction can have fluctuating levels of VWF. When these dogs are given thyroid supplementation, VWF levels can increase to within normal limits, which could prevent accurate diagnosis of their genetic status for VWD. For example, carriers of VWD might test as normal when on thyroid medication. Because of the apparent increase of the bleeding tendency in dogs with VWD and hypothyroidism, breeders and veterinarians must be aware of the increased risk and be more cautious about breeding or performing surgery on dogs with both problems.
GENETICS AND RECOMMENDATIONS TO BREEDERS
One form of VWD is inherited as an autosomal dominant trait which can reveal itself with varying physical signs. Or, in other words, the dog can have a differing levels of the VWD gene. This type of inheritance is termed 'incompletely dominant.' Both sexes can be equally affected in VWD, unlike in hemophilia, which is an X-linked recessive trait that classically affects only males. While the dominant expression of the VWD gene produces reduced levels of VWF in homozygotes with a double dose of the gene, it also does so in heterozygotes with a single-gene dosage. Clinical expression of VWD as a bleeding tendency, however, is inherited in the two forms mentioned at the beginning of this article. The common form is an autosomal incompletely dominant condition in which homozygosity is usually lethal, and heterozygotes can be either carriers with no physical symptoms, or affected to a varying degree. The more rare form of VWD is an autosomal recessive disorder in which clinically affected individuals are homozygous for the trait and express a double dose of the gene, one from each heterozygous, asymptomatic parent.
The following possibilities occur when animals with the VWD gene are mated:
If a normal dog, free of the VWD gene, is mated to one affected with or carrying the gene, about half of the litter will be affected or will be carriers, like the VWD parent, and the other half will be normal, free of VWD.
If two affected animals or two carriers are mated, three-quarters of the puppies will have the VWD gene. One quarter of the litter will be severely affected (perhaps fatally at birth), one-half will be affected or carriers like the parents, and the remaining one-quarter will be normal.
The more severely affected the parent, the more likely that it will produce severely affected bleeder puppies. Thus, a VWD carrier, which is clinically normal but transmits the VWD gene (trait) to some of its progeny, is the least likely to produce a clinically affected bleeder pup, provided the mate was VWD free. Mating two carriers of VWD, however, doubles the VWD gene, and puppies that get a double dose (one gene from each parent) are more severely affected than either parent. This is obviously an undesirable situation.
The following recommendations are offered to reduce the prevalence of VWD in a population: Blood tests for VWD should be taken in animals that are related to those bloodlines known to have the problem, as well as other top-producing or winning foundation stock. Pedigrees can be sent to the New York State Department of Health for evaluation of whether the animals or bloodlines are closely, distantly or not related to those of known affected families. Ideally, affected animals and carriers of VWD should not be bred. However, it may not be feasible for a breeder to eliminate these dogs as breeders, especially if they are desirable for other reasons. Therefore, if breeding affected animals is necessary to preserve important bloodlines or type, the following should be considered:
Breed asymptomatic carriers to normal dogs and blood test the pups. Half of the litter should be normal and the other half should be carriers. This is the best alternative to preserve bloodlines.
Mate two asymptomatic carriers only with caution and blood test the pups. Remember that only one-fourth of the litter will be normal and another one-fourth will be more severely affected than either parent.
Breed mildly or moderately affected animals only if essential to the breed and then breed the affected dog only to a normal animal. Be sure to blood test the pups, since half of the litter should be normal, and the other half will be affected like the parent. We do not advise this type of mating.
Do not mate two affected animals.
Never breed a severely, clinically affected animal.
Diagnostic tests for canine VWD are similar to those used for human VWD. Specialized VWF assays are required because VWD is indicated in the blood by a deficiency or abnormality or one or more proteins of the factor VIII complex. Screening coagulation tests (APTT, PT and TCT) are nondiagnostic. A brief explanation of the factor VIII complex is given in the sidebar on the following page to help make the tests used to diagnose VWD easier to understand. Dogs clinically affected with VWD also have prolonged bleeding times, abnormal platelet retention in vitro, and variable factor VIII coagulant activity levels(normal to moderately reduced). Definitive diagnosis is by finding reduced levels or no levels of FVIIIR:Ag and/or the platelet-related assays of VWF (ristocetin cofactor). A practical way to distinguish between asymptomatic heterzygotes and heterozygotes clinically affected with VWD (both have reduced levels of FVIIIR:Ag) is to determine the cuticle bleeding time. This test is performed by laying the dog on his side and cutting one or more toe nails too short with a standard, guillotine-type (Resco) nail clipper. With the foot undisturbed, the bleeding should cease within five minutes in normal dogs. Dogs with hemostatic defects, such as VWD or platelet dysfunction, have prolonged initial bleeding times (some never stop bleeding and must be cauterized) or begin bleeding again after initial clotting. A convenient way to perform this test is as a presurgical screen once the animal is anesthetized and the surgical site is being prepared. Clinical experience with this technic as a presurgical screen has been useful in identifying dogs that may bleed excessively at surgery.
GENETIC SCREENING FOR VWD
Screening for genetic defects has been used successfully in people for many years and more recently has been applied to animals. In the mid-1960's, veterinarians began screening dog populations for inherited eye diseases and hip dysplasia. Eventually, organizations were developed to register animals free of the major hereditary conditions known to affect purebred dogs. In New Zealand and Australia, mass screening of cattle for mannosidosis has been used effectively since the mid-1970's to control this devastating disease. The common practice of line breeding and inbreeding purebred dogs facilitates both the transmission and recognition of all types of genetic defects. Also, because companion animals live in close daily contact with their owners, illnesses are more likely to be noticed and treated.
The screening program developed in our laboratory for inherited bleeding disorders of purebred dogs evolved from an ongoing consultation and referral practice in veterinary hematology. The number of dogs screened for VWD by blood testing has increased steadily from the inception of our program in 1976 through 1980. Since 1980, the number has stabilized; this reflects implementation of planned matings between normal and carrier stock to eliminate the gene once heterozygotes have been identified.
In developing an accurate test to detect heterozygotes, we found, as in bovine mannosidosis, a skewed distribution between normal and abnormal populations, with a small but defined area of overlap. Despite this overlap, we have developed an effective program to identify heterozygotes and to gradually eliminate them from the breeding population. Data in support of this conclusion have shown a significant decrease in the overall prevalence of VWD in Scottish Terriers, Golden Retrievers and Miniature Schnauzers. The prevalence of VWD has been reduced in Scotties from 35% to 11%, in Goldens from 15% to 6%, and in Schnauzers from 25% to 18%. The prevalence in the other commonly affected breeds is essentially unchanged. This probably reflects a combination of insufficient numbers tested to implement planned matings for control of the gene and/or the need to increase breeder and veterinary awareness and acceptance of the importance of VWD.
The accuracy of the testing program in detecting the VWD genotype has been evaluated by retrospective analysis of the results to date for all three mating types (normal x normal, normal x carrier, carrier x carrier). The rate of misclassification of genetic status by this test is only 2-3%, which means that in 97% or more cases, the test is a reliable predictor for genotype. Cumulative data from these studies are currently being entered into a newly developed computer program to facilitate subsequent analyses.
FACTOR VIII COMPLEX
Factor VIII circulated in plasma as a complex of two proteins: the factor VIII-coagulant activity protein, which is severely deficient in hemophilia A and often is reduced in severe forms of VWD; and the VWF protein (also called after factor VIII-related protein), which has several important biologic properties that control bleeding and is deficient or abnormal in VWD but normal in hemophilia. Thus, one can readily distinguish between VWD and hemophilia even without a family history to establish the inheritance pattern, because animals with VWD have abnormal or low levels of VWF protein and hemophiliacs do not.
One of the properties of the VWF protein is its ability to cross-react in immunologic tests with antibodies formed against it in other species. This immunologic property of VWF protein is called factor VIII-related antigen (FVIIIR:Ag) and is measured routinely in plasma by the Laurell electroimmunoassay. Our screening program is based on this technic, using antibodies formed against VWF protein purified from normal canine plasma. (Such antibodies are not commercially available for animals, and cross-reactivity with readily available human antibodies varies among species, has less avidity, or is absent.)
The sensitivity, or lower limit of detection of this technic is about 7%. Thus, levels of 7% of normal or less of this plasma protein are undetectable (zero) in the assay. However, by using a more sensitive research technic that detects as little as 0.1% of the antigen, dogs affected with VWD can be classified into two groups. Scottish Terriers and Chesapeake Bay Retrievers that are homozygous for the autosomal recessive form of VWD have no detectable antigen and are truly zero level animals, whereas their heterozygous (carrier) parents and those with the other form of VWD (incompletely dominant expression) have reduced but measurable amounts of antigen.
To establish the normal range for canine FVIIIR:Ag, we measured levels in plasma from over 125 healthy purebred dogs of breeds in which VWD has yet to be recognized. The antigen levels in these animals ranged from 60-72% of normal, with a mean value of 93 ± 33%.
MANAGEMENT AND TREATMENT: GENERAL CONSIDERATIONS
Patients with bleeding disorders cannot be properly treated without an appropriate physiologic and physical environment for hemostasis, tissue repair and prevention of recurrence. An extremely important aspect of medical management is avoiding use of drugs known to interfere with hemostasis. For example, aspirin, phenylbutazone, phenothiazine tranquilizers, estrogens, plasma expanders (Dextran, HES), nitrofurans, sulfonamides, antiinflammatory drugs, penicillins, and local anesthetics. These are contraindicated for patients with moderate or severe hemostatic defects, since they impair platelet function and further compromise the stability of the hemostatic plug.
Any live-virus vaccine or viral infection can impair platelet and/or endothelial cell production and turn over. The effect occurs during the viremic phase after vaccination or exposure (usually at 5-10 days) and results in relative thrombocytopenia or endothelial injury, which may prolong bleeding time and predispose the animal to hemorrhagic problems. Platelet reductions of 100,000/yl can occur. During this period, animals with hemostatic defects are at risk and should be evaluated carefully for signs of bleeding. Elective surgical procedures, such as ear cropping, ovariohysterectomy, castration and dental surgery, should be performed within 48 hours after vaccination or should be postponed for 10-14 days. In most cases, animals admitted for elective surgery are vaccinated immediately or within 24 hours of surgery, which accounts for the relatively few vaccine related bleeding problems.
As mentioned earlier, hypothyroidism produces a bleeding tendency and exacerbates the clinical expression of concomitant VWD. Thus, asymptomatic heterozygotes for VWD can exhibit a bleeding tendency if they also become hypothyroid. This is a common situation in the Doberman Pinscher; many of these dogs have very low levels of T4 and/or T3, with abnormal TSH response tests, yet show none of the typical overt signs of thyroid disease. Hypothyroid animals with concomitant VWD usually show slightly to moderately reduced platelet counts (70,000-100,000/yl) and very long cuticle bleeding times, in addition to their low VWF activity.
Recent clinical experience with Dobermans admitted for bleeding episodes, such as nosebleeds or severe hematuria, has shown that thyroid supplementation alone reduces and then controls the bleeding within 24-48 hours. This finding supports our unpublished experimental studies that showed that when treated with T4, seven Dobermans with VWD and mild hypothyroidism had a two- or three-fold increase in their baseline levels of VWF activities within 24 hours. This peaked at three days and was sustained for another three to four days. The standard therapeutic dosage, based on body weight as recommended by the manufacturer, should be used and the animal should have a pretreatment serum specimen collected for resting T4 and T3 determinations. Follow-up of Dobermans treated with thyroid medication to control bleeding has shown that most were severely hypothyroid and the remainder were mildly abnormal or borderline normal. Therefore, our recommendation for all Dobermans with serious bleeding is to collect a serum specimen for thyroid and VWD measurements and then treat with thyroid replacement hormones pending results of blood tests.
Topical Treatment: Microcrystalline collagen (Avitene: Alcon), a topical hemostat, is superior hemostatically to pressure alone and/or oxidized cellulose cloth (Oxycel: Parke-Davis) or thrombin-soaked gelatin sponge (Gelfoam: Upjohn). Thrombin itself also has been used as a topical agent to control bleeding.
Blood or Blood-Component Replacement: Details for the type and volume of blood or blood products recommended to treat moderate or severe bleeding disorders are discussed elsewhere. The preferred anticoagulants for collection of whole blood for transfusion are acid-citrate-dextrose or citrate phosphate dextrose. Use of heparin is not recommended because it activates platelets, causing them to clump. Blood plasma products used to control and treat bleeding should be as fresh as possible or fresh-frozen, because coagulation factors and platelets are labile. As mentioned above, animals with bleeding disorders are likely to require repeated transfusions during their lifetimes and thus are at risk for transfusion incompatibilities. Use of unmatched whole blood, therefore, is contraindicated except in life-threatening emergencies. Appropriate therapy involved IV infusion of fresh universal donor or cross-matched blood at 3-5 ml per pound of body weight. The treatment of choice for VWD, when RBC's are not required, is canine plasma factor VIII concentrates (cryoprecipitates), but these are not commercially nor readily available for animals. An alternative is to give fresh-frozen, homologous plasma at 3-5 ml per pound of body weight once or twice daily.
Because platelets and coagulation factors have relatively short in vivo lives, control of bleeding episodes requires that the daily amount be divided and given as two regularly spaced transfusions. This regimen also reduces the risk of circulatory overload. For elective or other surgery, clinically affected VWD patients should receive fresh, compatible whole blood, at 3-5 ml/lb, two to four hours beforehand. The ability of the transfused
VWF to reduce the bleeding time lasts for up to four hours, whereas the other properties of the factor VIII complex remain active for 10-24 hours. For surgery on a VWD heterozygote with an unknown or no bleeding history, the cuticle bleeding time should be performed as a presurgical screen. If the time is within five to six minutes (normal), this does not preclude the existence of a mild underlying hemostatic defect but indicates that the surgeon probably will not encounter significant bleeding.
Instructions for Preparation
of von Willebrand's Disease Samples
Fill a blue-top Vacu-Tainer to capacity, mix well and centrifuge at 2500-3000 rpm for 15 minutes. Aspirate the supernatant plasma with a plastic pipette, siliconized pipette or a small plastic syringe, such as a tuberculin syringe. Place the plasma into a small plastic tube and freeze. Ship within 1-2 weeks after drawing.
All samples should be frozen initially and sent on cold pack to the laboratory. The sample may thaw in transit but samples in shipment longer than three days may have slightly reduced values. Dry ice is not necessary, but samples should arrive in a cool condition. Shipping by regular mail is quite satisfactory if the above criteria are met (cool, in three days or less).
Test Restrictions For Genetic Screening of Healthy Dogs
Do not test unhealthy animals or animals receiving any type of medication for recent illness or vaccination within the previous 14 days. If the animal is being given long-term medication or heartworm preventive, please indicate type and duration (e.g., Caricide or thyroid medication). A fasting blood sample is not necessary.
Tests with borderline findings between "normal" and "suspect" carriers will be repeated. Therefore, in these cases, additional time will be required to repeat the test. Those with <60% FVIIIR:Ag activity are normal, those with 40-60% are carriers, and those with less than 40% are considered FVIIIR:Ag deficient.
* These instructions are for specimens to be submitted to the veterinary Reference Laboratory for analysis. Readers should consult with their local diagnostic laboratory for instructions if the veterinary Reference Laboratory is not used.
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Original Doc: vwd02.doc
Update On Von Willebrand’s Disease In Scottish Terriers With Emphasis On The Acquired Disorder
W. Jean Dodds, DVM
Wadsworth Center for Laboratories & Research
New York State Department of Health
P.O. Box 509
Albany, NY 12201-0509*
Scottish terrier fanciers have contacted us recently requesting information about the influence of autoimmune diseases and thyroid disease in particular upon the level of von Willebrand factor (vWF) and the expression of von Willebrand’s disease (vWD) in the breed. Questions and concerns have been raised about some recent vWD test results because owners were expecting to have normal testing offspring from parents that have tested normal or came from normal testing ancestry. Some puppies from these litters were coming up with results at the lower end of normal range (60-69% vWF antigen, vWF:Ag), borderline (50-59%), or even an occasional one testing below 50% vWF:Ag. How could this occur?
In order to help explain the various factors that could pertain here, we thought an article about the variables affecting vWF production and the influence of autoimmune disease on its regulation and clinical expression would be helpful.
Influence of Sample Collection, Processing and Transport
Errors or difficulties encountered when collecting and processing blood samples for vWD screening still remain the most common reason for inconsistent results. We realize that its not always easy to obtain proper samples with clean venipuncture and good blood flow rate, especially with puppies or breeds that like to express their displeasure at having to endure this indignity! Samples containing significant hemolysis (red blood cell breakdown) or clots, even small ones, indicate that the animal was stressed and/or the sample was not obtained cleanly. Results for vWF:Ag on these samples may be artificially raised, lowered, or invalid. We routinely examine the quality of each sample, note any unusual findings on the written report, and advise you to retest the animal if indicated. We realize that this is more easily said than done. However, in reviewing records of earlier tests when future generations appear to be testing lower than expected, we quite often see that original results of questionable validity were never confirmed by a retest. So one way to assure more consistent interpretation of vWD genotype for a breeding program is to retest all foundation stock if there is or was any question about the sample quality on an original test. Having more than one test result gives you more confidence about the true reading of your stud dog or brood bitch. As expected, animals with normal levels are rarely retested whereas those with low levels are rechecked one or more times. Unfortunately, the higher readings on rechecks are often the ones that breeders remember or elect to proceed with -- which is only human nature, but may not reflect the animal’s true status. If there was no apparent reason to obviate one or more of the test results, then a prudent approach would be to consider averaging all values or at least keeping in mind that some other factor(s) might be influencing vWF levels in this animal.
When transporting samples to us for testing they should stay cold or frozen in transit; an
*Home: 938 Stanford Street, Santa Monica, CA 90403 [(213) 828-4804].
overnight mail or package express system is preferred over regular mail. Samples should be placed in an insulated container with a frozen cold pack even in wintertime, because during transport the box could be kept indoors near a heating unit. Sending the samples directly from your location to us is preferable to having a commercial laboratory pick up the specimen at your veterinarian’s clinic and ship it to one location where it is then reshipped to us or another place for testing. The fewer times a sample has to be reshipped prior to analysis, the less chance for transit artifacts affecting results. In fact if samples become heated or otherwise altered by shipment, levels of vWF:Ag often go up. The owners may be pleased to obtain high testing results but the actual level could be lower. (as an example, we invalidate all results above 200% vWF:Ag.)
Disparity Between Duplicate or Repeat Test Results from Different Laboratories
On several occasions, fanciers have indicated that they split samples or sent two samples within a few days to our laboratory with different animal identifiers, or have sent them to us and another university or commercial laboratory at the same time. The purpose has been to check the reliability and validity of the testing process. We have no problem with anyone wishing to do so because it is the cornerstone of proper quality control. If a problem exists in our testing procedure we want to hear about it in order to investigate the situation. If you do this, however, please let us know afterwards so we can correct our records when duplicate samples were submitted under different names.
The assay we run routinely is basically the same as its been for the past 10 or more years. We have recently developed a newer, more sensitive and equally reliable test that we’ve been running in parallel with the traditional assay. So far results of the two tests have been remarkably consistent. The same supervisory laboratory staff has coordinated and reviewed our vWD testing results for this entire period! We have very strictly standardized quality control procedures and the reproducibility of the assay is + 3% with a coefficient of variation of less than 9%. While any system can experience an occasional human error, including our own, the error factor is minimized by our quality control/review procedure.
A group of commercial veterinary testing laboratories offer vWD testing now. Most of these laboratories send their samples directly to us but a few do their own testing in-house. If you use this system through your veterinarian, find out how or where the laboratory does the test. A few human clinical laboratories also offer veterinary testing. Be cautious here because if the routinely used human assay has not been adapted for use in the dog, the results may be erroneous. Secondly, if a veterinary laboratory performs the test in-house using our reagents their staff may be less aware of the need to check sample type and quality and note any unusual findings on the report. We know that this situation occurs because of healthy animals that received test reports of < 7% vWF:Ag when the specimen submitted turned out to be serum (already clotted and so no vWF:Ag was left) rather than plasma.
The most frequent problem encountered with divergent results between so-called “duplicates” is that the specimens are not truly duplicates. For example, the ideal way to collect true duplicates is to preload an empty syringe with the correct amount of trisodium citrate anticoagulant to achieve a final concentration of 1 part citrate to 9 parts blood (1 in 10 final dilution). After collecting a free-flowing sample from a clean venipuncture, the sample is mixed and transferred to a test tube for centrifugation. After centrifugation, the plasma at the top of the tube is removed, placed into another empty test-tube, mixed thoroughly and then half of it is transferred to a third empty test tube. Only in this manner are the two specimens equivalent. The mistakes most commonly made in trying to send duplicates are taking two separate samples from different veins one after the other (the animal is more stressed for the second one), or the plasma is incorrectly divided after spinning the blood down because the top part is removed and put in one tube and the bottom part is placed in the second tube. As plasma proteins centrifuge according to weight and mass, the heavier proteins are at the bottom of the plasma and lighter ones at the top; this does not produce true duplicate samples.
Finally, one other common problem occurs with the use of VacutainersR to collect the sample, if they’re not used as originally intended. In our experience many veterinary clinics collect blood first into a dry, empty syringe and then add it to the VacutainerR containing the citrate anticoagulant, instead of drawing the blood directly through the needle by vacuum into the tube. This modification allows for clotting to begin before the anticoagulation occurs and so samples are partially clotted and will most likely have lower readings for vWF:Ag.
Inherited and Acquired vWD
vWD in humans and dogs is now recognized to be congenital (present at birth) and inherited and/or acquired secondary to familial autoimmune thyroid disease. Presently, 44 of 53 dog breeds known to have vWD also transmit familial hypothyroidism. The Scottish terrier is one of the breeds having both disorders. While the prevalence of thyroid disease has increased rapidly over the last decade within Scottish terrier stock, vWD has declined until recently through the collective efforts of breeders to test and screen out carriers from their breeding programs.
1. Inherited (Congenital) vWD
In Scottish terriers, vWD is an autosomal recessive disorder classified as Type III vWD. Clinically affected animals are homozygous for the vWD gene and have two asymptomatic, heterozygous (carrier) parents. The summary and recommendations about vWD’s clinical and gene expression as described in the Scottish Terrier Association’s Handbook are still correct.
To review, vWD in Scottish terriers expresses a mild to severe bleeding diathesis that usually involves mucosal surfaces and is exacerbated by physical, emotional and physiological stresses as well as by other concurrent diseases. Typical clinical signs include: recurrent gastrointestinal hemorrhage with or without diarrhea; recurrent hematuria; nosebleeds; gingival, vaginal, and penile bleeding; lameness that mimics eosinophilic panosteitis; stillbirths or neonatal deaths (“fading pups”) with evidence of bleeding at necropsy; prolonged estrual or postpartum bleeding; hematoma formation on the surface of the body, limbs, or head; excessive umbilical cord bleeding at birth, and excessive bleeding from toe nails cut too short or after dewclaw removal. Affected dogs may bleed to death from surgical procedures. Diagnostic tests require specialized vWF assays. Screening coagulation tests (APTT, PT and TCT) are nondiagnostic. Animals affected with the recessive Type III disease are homozgotes, have long bleeding times and zero vWF:Ag, whereas their heterozygous parents have reduced levels (15-60% of normal) and have normal bleeding times unless some other hemostatic problem coexists.
2. Acquired VWD
In dogs, vWD is exacerbated by concurrent hypothyroidism, so that asymptomatic carriers of vWD may exhibit a bleeding tendency if they subsequently become hypothyroid, a common situation found in many breeds including Scottish terriers. The concomitant prevalence of vWD and hypothyroidism confirms the link between the synthesis and/or metabolic regulation of thyroid hormones and vWF. Furthermore, hypothyroid dogs may exhibit low platelet counts (thrombocytopenia) and associated mucosal surface bleeding. In humans, a bleeding tendency may be an early manifestation of hypothyroidism. This includes easy bruising and thrombocytopenia, abnormal platelet function which returns to normal after treatment with thyroid hormone, and acquired vWD similar to the common Type I form of this disorder (low VWF:Ag). It is generally impossible to distinguish between the inherited and acquired types of vWD in an individual patient with currently available techniques.
Circulating antithyroid antibodies are often present in humans and dogs with autoimmune thyroid disease (called thyroiditis or Hashimoto’s thyroiditis) several years before the thyroid disease becomes clinically apparent. Thus animals with thyroid dysfunction can have fluctuating levels of vWF and, when placed on thyroid supplementation, levels can increase to within normal limits, which could preclude the accurate diagnosis of their genetic status for vWD (i.e., carriers of vWD might test as normal when on thyroid medication). Because of this apparent exacerbation of bleeding tendencies in dogs with vWD and hypothyroidism, breeders and veterinarians need to be aware of the increase risk and be more cautious about breeding or performing surgery on dogs with both problems.
In humans, antithyroid antibodies can be transferred from mothers with Hashimoto’s thyroiditis to their infants in breast milk. Neonatal hypothyroidism can occur and is picked up on the routine newborn screening programs run by state and local public health agencies. Whether a parallel transfer of maternal antithyroid antibody occurs via the colostral milk to newborn puppies is unknown but is likely. What effect, if any, the transfer of these antibodies could have on the development and health of puppies is also unknown . Both humoral and cell-mediated immunity could be affected, especially when growth hormone and thyroxine modulate the thymus and thereby immune function. Perhaps the presence of maternal circulating antithyroid antibodies during the first several months of life could alter immune recognition or development sufficiently to render these puppies more susceptible to other immune-mediated stressors (viruses, vaccines, drugs, toxins, etc.). These antibodies could play a role in stimulating the active immunological process that leads to the development of immune thyroiditis.
3. Magement and Treatment
The severely limited availability of blood products for clinical use in veterinary medicine has necessitated the development of alternate strategies for the treatment and management of bleeding disorders. In those breeds like Scottish terriers in which both vWD and hypothyroidism occur relatively often, the administration of oral thyroid hormone has been effective in controlling bleeding episodes exacerbated by these two disease. Clinical signs of bleeding are lessened or controlled within 48 hours after therapy is initiated. In man, L-thyroxine therapy has been shown to reduce levels of circulating antibodies responsible for thyroiditis and hypothyroidism. Presumably this plays a role in the reversal of the bleeding tendency associated with thyroid disease. In dogs, the toenail bleeding time is dramatically shortened and in some cases may even be corrected to within normal limits (<5 mins) from pre-treatment values beyond 20 minutes. Thus, thyroid supplementation alone may suffice to control bleeding in mild to moderate vWD, a situation analogous to the use of desmopressin (DDAVP) or danazol to control bleeding in man. DDAVP treatment has recently been shown to improve vWF levels in normal dogs and dogs with vWD, although relatively high doses are required and the response is transient (1-3 hours); refractoriness to this treatment can develop rapidly.
The standard dosage of L-thyroxine for treatment of vWD [enhances platelet stickiness (adhesion) and improves vWF activity] is 0.1 mg/10 lb. body weight given twice daily. The therapeutic response should be monitored with serial toenail or other bleeding times and periodically by thyroid function tests; dosages of medication should be adjusted accordingly to maintain thyroid levels (total T4, free T4, total T3) within the upper half to third of the adult normal ranges.
Production of vWF and Causes of Autoimmune Thyroid Disease
The endothelial cells lining blood vessels are the primary and essentially sole source of vWF production in the dog. Thyroid dysfunction from familial autoimmune thyroiditis is by far the most commonly recognized cause of acquired vWD in dogs. Production and/or secretion of vWF from endothelial cells is clearly altered by thyroiditis. The most likely cause of lower than expected vWF:Ag scores on dogs with parents known to be free of the vWD gene is therefore this thyroid disorder, whether or not it is expressed to date, because the pathologic process associated with autoimmune antithyroid antibody production can take several years to induce the end stage of clinical hypothyroidism. If you breed them, they will transmit susceptibility for the thyroid problems to some of their offspring.
Our cumulative data on many breeds affected by familial autoimmune thyroid disease, including Scottish terriers, indicate that reduction in vWF:Ag production associated with thyroiditis is becoming quite common. To repeat, the clinical and laboratory features of this acquired form of vWD are indistinguishable from the congenital inherited form of vWD. Thus while the inheritance and carrier detection program for vWD have not changed, the situation over the past decade and especially in the last 5 years is complicated by the rapid rise in the incidence of thyroid and other autoimmune disease in your breed.
This is illustrated by a summary of our latest statistics on the prevalence of vWD over the past 8 years.
The dramatic increase since 1987 in the number of dogs having abnormal levels of vWF:Ag is real because the number of dogs tested each year continues to be large enough for statistical validation. There are three possible explanations for this increase, which by the way has also occurred in the five other breeds that we test most often (Doberman pinchers, golden retrievers, standard poodles, Shetland sheepdogs, Pembroke Welsh corgis). The first envokes a shift in the measurement of vWF:Ag; this possibility is ruled out because our assay has been standardized internally both vertically and horizontally over time and so varies less that + 5% of the actual value. Secondly, the prevalence of inherited vWD is increasing because tested or untested carriers continue to be bred. Lastly, the increasing prevalence of thyroid disease has produced a parallel increase in the acquired vWD. The most likely explanation is a combination of the last two possibilities.
There are 4 primary causes of autoimmune disease and autoimmune endocrine (e.g., thyroid ) disease in particular: a) genetic predisposition; b) viral infection or exposure; c) hormonal influences especially of sex hormones; and d) stress. In humans with a parallel form of thyroid disease (Hashimoto’s thyroiditis or lymphocytic thyroiditis), they have identified a specific profile of the major histocompatability complex, HLA-DR, which conveys genetic susceptibility to the disease. Furthermore, in May 1989 a research team in England discovered a novel retrovirus, distinct from but related to the HIV virus that transmits AIDS, that is associated with human thyroiditis. The bottom line is that viruses capable of inducing immune dysregulation in genetically susceptible stock can initiate autoimmune thyroid disease under the appropriate environmental conditions (e.g., hormonal imbalance, stress, drug or toxin exposure, dietary factors, etc.). There is every reason to believe that the canine disease has similar causation. In the past several months, three veterinary groups have demonstrated reverse transcriptase activity ( a result of retrovirus exposure) in canine lymphoma/leukemia cell lines. Dogs and especially closely related linebred/inbred purebreds have the genetic makeup that conveys susceptibility, the viral elements are there, and reproductive hormonal and stress influences are prevalent when one breeds for performance, etc. The occurrence of low levels of vWF:Ag and the increased risk of bleeding it conveys from acquired vWD is one of several major undesirable consequences of this common autoimmune endocrine disease.
We believe it is very important for Scottish terrier fanciers to understand the influence autoimmune thyroid disease has on vWF levels and the expression of acquired vWD. This is a more complex and important problem which impacts the survival and vigor of the Scottish terrier breed than is the Type III autosomal recessive form of vWD which an be controlled relatively easily with an open, honest testing and planned breeding program.
About 90% of thyroid abnormalities in the dog have an inherited autoimmune basis. Linked to these disorders are other immune-mediated problems such as autoimmune hemolytic anemia and thrombocytopenia; bone marrow failure; leukemia and lymphoma; systemic lupus erythematosus; seizure disorders; chronic infections; immunosuppressive viral infections like distemper, parvovirus and retrovirus diseases; chronic active hepatitis (liver disease); immune kidney and adrenal (Addison’s) diseases; chronic allergic and immune skin and muscle disorders.
Thyroiditis is the immune-mediated process characterized by the presence of antithyroid antibodies in the blood or tissues. This condition usually progresses eventually to thyroid disease. Antithyroid antibodies can be present in euthyroid (normal), hypothyroid, or hyperthyroid humans or animals, i.e., can be present while a dog is still testing normal for thyroid function, and can be doing things to other tissues such as bone marrow, fetuses, and other fast-growing cells. Part of the management of autoimmune disorders is to test for the presence of thyroid dysfunction and antithyroid antibodies and to treat with thyroid hormones to inhibit the process. Therefore, we need a way to diagnose the presence of antibodies before the disease develops.
A typical thyroid test measures the total presence of the T4 hormone in the body. But an important measurement is the small fraction of the total T4 which is not bound in the body and is therefore available to the tissues. This “free” T4 is converted in the tissues to T3 which is important for intracellular function. In the newer thyroid profiles available at specialized veterinary laboratories in the US and Canada, they test for total T4, total T3, free T4, cholesterol, and free T3. Cholesterol should be measured from the same blood sample as the thyroid tests. In dogs with thyroid dysfunction the higher the cholesterol level, the lower the level of free T4. Owners can obtain a printout listing all the test results for their dog, as well as the “K” value, which is a linear constant between Free T4 and cholesterol.
With respect to K values, those which are above +1 and preferable above +5 are considered to be normal. Less than -4 indicates an animal with primary thyroid disease, even if there are no overt symptoms. Values between -4 and +1 are either early thyroid disease and/or a nonthyroidal illness (another illness which depresses thyroid levels). Thyroid deficiency even at early subclinical stages can cause irreversable damage in the body, and the value of this more sensitive test is that problems can be detected before damage has become so severe that clear visible symptoms are present.
For a dog displaying symptoms of autoimmune disease, the K value can be helpful to indicate whether the dog has low, normal or high thyroid function, and if the autoimmune problems are likely to be responsive to thyroid hormone supplementation. If test results are normal, and the dog is nevertheless ill, proceed to do other diagnostic tests such as that for antithyroid antibodies [antithyroglobulin, anti T4 and anti T3 autoantibodies].
Michigan State University has recently introduced antithyroid hormone antibody (anti T4 and anti T3) testing as part of their complete thyroid panel. This is an important step towards more effective thyroid testing. Contact the Endocrinology Section, Animal Health Diagnostic Laboratory, P.O. Box 30076, Lansing, MI 48909 (517-353-0621). Our laboratory and the Veterinary College at University of Florida, Gainesville, offer antithyrogobulin antibody testing.
These thyroid panels and antibody tests can also be used for genetic screening of apparently healthy animals to evaluate their fitness for breeding. A bitch with antithyroid antibodies in her blood may pass these along to her puppies in her colostral milk. Also any dog having circulating antithyroid antibodies can eventually develop clinical symptoms of thyroid or other autoimmune diseases. Therefore thyroid screening can be very important for potential breeding stock.
Thyroid testing for genetic screening purposes is less likely to be meaningful before puberty. Healthy young dogs (less than 15-18 months of age) should have thyroid baseline levels for all parameters in the upper third of the adult normal ranges. In fact, for optimum thyroid function in screening breeding stock, levels should be at least at the mid-point of the so-called “normal” ranges because lower levels may well be indicative of the early stages of thyroiditis among relatives of dog families known to have thyroid disease.
When treating with thyroid hormones: T4 is very forgiving, T3 can be easily toxic. With T4, dosage must be twice per day, as about half of it is metabolized and excreted from the body within 12 hours. For follow-up testing 4 to 6 weeks after initiating treatment, test at 4 to 6 hours after the morning dosage. For optimum levels, dogs should test in the upper third of the normal ranges after 4-6 weeks of therapy.
Dogs on long-term supplementation with thyroid hormones, should be monitored with complete panels on a regular basis (every 6 to 12 months), and dosages should be adjusted accordingly.
For Clubs Holding Joint Blood Testing for vWD or Other Bleeding Diseases and ThyroidDysfunction
Blood samples should be drawn for the coagulation testing (vWD or other tests) first because anticoagulated specimens are needed from unstressed animals to optimize valid results.
A separate sample of clotted blood to obtain serum is required for thyroid function profiling.
We would appreciate your assistance in interpreting the test results from your dog(s). Please indicate when submitting samples from your animals whether they have any type of health problem or family history of liver and/or thyroid disease. If your dog(s) is completely healthy at the time of testing, please also indicate this fact so that we can avoid assumptions that may be incorrect.
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