Blood Platelets in Cavaliers:
Too Few (Thrombocytopenia) and
Too Large (Macrothrombocytopenia)
Are Usually Not a Serious Health Disorder


IN SHORT

Up to half of all cavalier King Charles spaniels may have both an abnormally low number of blood platelets and oversized platelets. Despite the low platelet counts, the typical cavalier's blood platelets function normally, and the dog does not appear to experience any health problems due to either the size or fewer numbers of its platelets. There are, however, exceptions to this typical situation.

Excessively low platelet counts normally is a sign which tends to alarm general practice veterinarians, and so it is vitally important that cavalier owners alert their vets about this benign condition in the breed when blood tests are ordered. More...

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IN DEPTH

As many as half of all cavalier King Charles spaniels may* have a congenital blood disorder called idiopathic asymptomatic thrombocytopenia, an abnormally low number of blood platelets, according to several recent studies. Blood platelets (also called thrombocytes) are disk-shaped blood elements which aid in blood clotting. Excessively low numbers are the most common cause of bleeding disorders in dogs.

* We say "may" because a 2008 Swedish study has found no thrombocytopenia in 27 CKCS when their blood was tested on a new device using the plateletcrit method which measures platelet volume.

CKCS giant platelet on blood smear (Univ. Minn. 2013)The platelets in the blood of many cavalier King Charles spaniels are a combination of those of normal size for dogs and others that are abnormally oversized. Cavaliers' oversized platelets are called macrothrombocytes. (See oversized CKCS platelet in blood smear at right.*) Macrothrombocytosis also is a congenital abnormality found in at least a third of CKCSs.

*Courtesy, Jessica Anne Kelley master's thesis, Univ. Minn. 

These large platelets function normally, and the typical cavalier does not appear to experience any health problems due to either the size or fewer numbers of its platelets. There are, however, exceptions to this typical situation. Most recently, a June 2010 study has confirmed that "The data supports previous findings that CKCS with macrothrombocytopenia have a normal coagulation response despite low platelet counts."

Nevertheless, researchers hypothesize that cavaliers' giant platelets are the source of high levels of serotonin which may contribute to mitral valve disease in the breed. Read the details here.

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Research Studies' Findings

-- platelet size and counting issues

Cavalier Bath TimeA 2002 Danish study report (Drs. Henrik D. Pedersen, Jens Häggström, Lisbeth H. Olsen, and others) concluded that cavaliers' oversized platelets could lead to erroneous diagnoses of thrombocytopenia when automated blood counters are used. An erroneous diagnosis of low platelet counts could result in unnecessary diagnostic tests for the cavaliers.

A 2005 Australian study (Drs. M. K. Singh and W. A. Lamb) of 152 cavalier King Charles spaniels, all from New South Wales, found that the platelet count of the cavaliers, as a group, was only 32% that of the mixed breed control dogs, and that 30% of the CKCS in the study had macrothrombocytes. The Australian researchers concluded that the data supported an autosomal recessive inheritance pattern.

In a 2007 Italian study (Drs. W. Bertazzolo, S. Comazzi, L. Sesso, P. Scarpa, G. Ru, and S. Paltrinieri), the researchers compared platelet counts, platelet estimations and platelet parameters in 41 cavaliers measured by different methods -- impedance cell counter, laser cell counter and microscopic estimation -- commonly used in veterinary hospitals and commercial laboratories. Quantitative buffy coat analysis was performed on 17 of the samples, selected from cavaliers which had low platelet counts detected by the cell counters. CKCSs with macrothrombocytes had significantly lower counts on impedance cell counter (34.1% dogs below normal -- < 1003×109/l), laser cell counter (26.8%) and microscopic estimation (22.0%). The quantitative buffy coat analyser result was only 5.8% below normal.

In a 2008 study at Auburn University (Drs. B. Davis, M. Toivio-Kinnucan, S. Schuller, M.K. Boudreaux) determined that "a mutation in the gene encoding β1-tubulin correlated with macrothrombocytopenia in CKCS." They concluded that "this information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombocytopenic CKCS with concurrent mitral valve regurgitation."

The Auburn researchers since have devised a DNA test for macrothrombocytopenia. See details at Diagnosis below.

-- possible relationship with mitral valve disease

It has been shown in studies of humans that mitral valve regurgitation can cause a reduction in the quantity of platelets, as well as increase their size, and alter their aggregation. In a 2003 Danish study (Drs. I. Tarnow, A. T. Kristensen, H. Texel, L. H. Olsen, and H. D. Pedersen), the researchers found that CKCSs with mild, moderate, and severe mitral valve regurgitation have decreased platelet function. So, mitral valve disease may be the underlying cause of both thrombocytopenia and macrothrombocytopenia in the cavalier King Charles spaniel. However, in a 2002 US study of 42 CKCSs, the researchers reported that: "There was no association of thrombocytopenia with murmurs, and no association of abnormal platelet aggregation with murmurs or thrombocytopenia."

In the 2004 US study led by Drs. Sara M. Cowan and Rebecca Gompf of the University of Tennessee, thrombocytopenia was present in 51.43% of the 69 CKCSs in the study, and macrothrombocytes were present in one third of the cavaliers. Co-incidentally, 38% of the dogs in the US study had cardiac murmurs. The American researchers found platelet ultrastructure to be normal. They have identified the disorder as Benign Inherited Giant Platelet Disorder (IGPD). They wrote that "further studies are indicated to determine ... any association with mitral endocardiosis [MVD]."

In a January 2010 report, Dr. Mark A. Oyama of the University of Pennsylvania suggested a linkage between oversized platelets in cavaliers and their significantly higher serotonin concentrations than in other breeds of dogs.  Serotonin and transforming growth factor-β are believed to play major roles in transforming valvular interstitial cells (VIC) into a more active myofibroblast, which is an important component of MVD in cavaliers. Read here about Dr. Oyama's continued research of the link between CKCSs' giant platelets and MVD.

Platelets: Resting & ActivatedIn an April 2014 report, members of the Danish team, Drs. S.E. Cremer, A.T. Kristensen, M.J. Reimann, N.B. Eriksen, S.F. Petersen, C.B. Marschner, I. Tarnow, and L.H. Olsen joined with USA's Dr. Mark Oyama to report finding a link between high-level serotonin-binding and highly-activated blood platelets and mitral valve disease and oversized platelets in cavaliers. They studied the levels of serotonin-binding and of platlet activation in 61 CKCSs and found that cavaliers with moderate-severe MVD and severe MVD tended to have higher levels of platelet activation, and that highly-active platelets and high-level serotonin-binding was strongly associated with oversized platelets. They concluded that further investigation into the significance of serotonin-binding.

In a July 2014 report, the international team found that platelet serotonin was elevated in cavaliers compared to other breeds, and that left ventricular myocardial and mitral valve leaflet tissue in deceased MVD dogs was elevated compared to dogs which died without cardiac disease.

Some researchers have suggested that Epsiodic Falling Syndrome and Fly Catcher's Syndrome in cavaliers may be associated with the breed's form of thrombocytopenia. Drs. Jens Häggström and Clarence Kvart of Sweden have noted in a 1997 article that thromboembolic events in the cerebral circulation of blood may be involved in these syndromes.

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Platelet Counting Procedures

-- automated and manual methods

CavalierHealth.org Copyright © 2004 Blenheim CompanyMost commercial laboratories use an automated counting system for blood cells, which determine cell types on the basis of their size and volume. Because cavaliers' platelets are so large, automated blood cell counters may not recognize platelets as being platelets and undercount them, thereby inaccurately lowering the platelet count. In a 2004 Danish study and the 2007 Italian study, the researchers found that all of their CKCS platelet counts using three different automated systems underestimated the actual counts determined manually. Antech Diagnostics, the largest veterinary diagnostic laboratory, specifically states on its website that "Platelets in this breed should be counted manually, because automated blood cell counters cannot distinguish the large platelets from erythrocytes and therefore underestimate the true platelet count."

An accurate platelet count can be obtained by visually counting the cells. Also, because the large platelets are so fragile, any blood samples should be extracted very carefully. Therefore, all blood samples from cavaliers should be taken in a very careful manner and preferably only from the dog's jugular vein, using a large bore needle, and then should be examined only under a microscope by an experienced clinical pathologist before making a diagnosis of low platelet count. The 2004 US study report recommends that the blood be anticoagulated with ethylenediamine-tetraacetic acid (EDTA) and citrate.

-- plateletcrit method

The days of mis-diagnosis of thrombocytopenia in cavaliers may be over!  IDEXX Laboratories has developed a hematology analyzer using fluorescent optics which measures the volume of platelets (called plateletcrit) in a blood sample.

This procedure has been found in a 2008 Swedish study to be superior to the customary manual and automated platelet counts for assessing the platelet status of cavaliers. The device is called the IDEXX VetAutoread Hematology Analyzer. In their report, the researchers, Harold Tvedten, Inger Lilliehöök, Anna Hillström, and Jens Häggström, reported:

"The major purpose of this study was to compare the QBC [IDEXX VetAutoread Hematology Analyzer] platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs. ... Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample.
"Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 109 platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum. Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass."

In view of the 2008 Swedish study's finding of no thrombocytopenia in any of the 27 cavaliers, could it be that the breed does not suffer from thrombocytopenia, but instead has been the victim of platelet counting methods which rely upon the number of platelets rather than their overall volume?  Macrothrombocytosis -- oversized platelets -- still were found in this study.  Researchers in the 2008 study found that the platelet volume is more important than the numbers for the function of the platelets.

In a 2012 study, the same team of Swedish researchers compared the results from IDEXX's VetAutoread hematology analyzer with a Bayer Diagnositics' device, the Advia 2120 plateletcrit analyzer, for 43 cavaliers and 15 other dogs. The results showed that Advia's platelcrit readings were lower than those found by the IDEXX device for cavaliers which had the lowest plateletcrits.

Similarly, in a January 2013 master's thesis, University of Minnesota student Jessica Anne Kelley studied blood tests of 139 CKCSs, to investigate the use of the plateletcrit as an assessment of total platelet biomass in cavaliers with macrothrombocytopenia. She concluded:

"In this particular model of an inherited asymptomatic macrothrombocytopenic platelet disorder, Cavalier King Charles Spaniels represent a group of individuals in which the bleeding risk is better evaluated using plateletcrit than platelet number. The platelet mass appears significantly lower in affected dogs when measured by count, but when measured by plateletcrit, more of the patients are appropriately within the reference range. Therefore, for the Cavalier King Charles Spaniels affected by this mutation, plateletcrit better represents the true total platelet biomass in peripheral circulation."
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Diagnosis -- DNA Testing

The typical CKCS with idiopathic asymptomatic thrombocytopenia does not appear to experience any health problems due to either the size or fewer numbers of its platelets. However, many veterinarians who are ignorant about the cavaliers' benign disorder hurriedly conclude that the dog is suffering from immune-mediated thrombocytopenia (IMT), which is a very serious autoimmune disease in which the dog's body attacks its own blood platelets as though they are a pathogenic bacteria or virus.

Petechiae on gumsIMT also has some very serious symptoms, such as an unusual susceptibility to bleeding (hemorrhaging), petechiae (small red or purple spots on the dog, caused by broken capillary blood vessels -- see photo at left), bruising, bleeding from the mouth or nose, pale gums, lethargy, weakness, increased respiratory rate, and/or dark, tarry stools that indicate the presence of blood in the GI tract. So, unless the CKCS also has any of those symptoms, it is extremely likely that all the cavalier has is the breed's benign platelet disorder.

DNAVeterinary researchers at Auburn University have devised a DNA test for CKCSs suspected of having oversized platelets. In their 2008 report, the research team identified a mutation in the gene that encodes beta1-tubulin, a protein involved in platelet production by megakaryocytes. Their DNA test is available to determine the presence or absence of this mutation. For more information about this DNA test, contact Dr. Mary K. Boudreaux at Auburn Univ., telephone 334-844-2692, email boudrmk@auburn.edu, or click here for the webpage.

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Treatment

No treatment is recommended unless the dog shows other symptoms of a blood-related disorder.

Cavaliers should not be treated for immune-mediated thrombocytopenia (IMT) as a precaution. Dog really suffering from IMT must be treated quickly, usually with intravenous doses of immunosuppressive drugs -- steroids, azathioprine, cyclosporine A and others -- to save the dog's life. Such treatments are severe and could do major damage to the healthy cavalier with nothing more than a low platelet count.

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Research News

July 2014: International team of cardiologists find cavaliers have higher serotonin concentrations in platelets. In a July 2014 study, cardiologist researchers from Denmark, the USA, and France (S.E. Cremer, G.E. Singletary, L.H. Olsen, K. Wallace, J. Häggström, I. Ljungvall, K. Höglund, C.A. Reynolds, N. Pizzinat, M.A. Oyama) compared serotonin concentrations in blood plasma of 26 cavaliers (12 healthy and 14 with mitral valve disease) and 19 non-CKCSs (8 healthy and 11 with MVD). They found that platelet serotonin was elevated in cavaliers compared to the other breeds. They also found that left ventricular myocardial and mitral valve leaflet tissue in deceased MVD dogs was elevated compared to dogs which died without cardiac disease.

May 2014: International team finds link between high-level serotonin-binding and highly-activated blood platelets and mitral valve disease and oversized platelets in cavaliers. Platelets: Resting & ActivatedIn an April 2014 report, a team of researchers from Denmark and the USA studied the levels of serotonin-binding and of platlet activation in 61 CKCSs. They found that cavaliers with moderate-severe MVD and severe MVD tended to have higher levels of platelet activation, and that highly-active platelets and high-level serotonin-binding was strongly associated with oversized platelets. They concluded that further investigation into the significance of serotonin-binding is warranted.

January 2013: University of Minnesota student's master's thesis confirms superiority of Advia's plateletcrit method. In a January 2013 master's thesis, University of Minnesota student Jessica Anne Kelley studied blood tests of 139 cavalier King Charles spaniels, to investigate the use of the plateletcrit as an assessment of total platelet biomass in cavaliers with asymptomatic congenital macrothrombocytopenia. She concluded:

"In this particular model of an inherited asymptomatic macrothrombocytopenic platelet disorder, Cavalier King Charles Spaniels represent a group of individuals in which the bleeding risk is better evaluated using plateletcrit than platelet number. The platelet mass appears significantly lower in affected dogs when measured by count, but when measured by plateletcrit, more of the patients are appropriately within the reference range. Therefore, for the Cavalier King Charles Spaniels affected by this mutation, plateletcrit better represents the true total platelet biomass in peripheral circulation."

See also, the January 2014 published version of this article, below.

September 2012: IDEXX's VetAutoread hematology analyzer tops Bayer Diagnostic's AdviaIDEXX 2120 in measurement of CKCS plateletcrits. Swedish researchers find that Bayer Diagnositics' Advia 2120 plateletcrit analysis of cavaliers was lower than expected, when compared to results from IDEXX's VetAutoread hematology analyzer. Read more here.

September 2011: Auburn Univ. offers DNA test for inherited macrothrombocytopenia in cavaliers. Veterinary researchers at Auburn University have devised a DNA test for CKCSs suspected of having oversized platelets. In their 2008 report, the research team identified a mutation in the gene that encodes beta1-tubulin, a protein involved in platelet production by megakaryocytes. Their DNA test is available to determine the presence or absence of this mutation. For more information about this DNA test, contact Dr. Mary K. Boudreaux at Auburn Univ., telephone 334-844-2692, email boudrmk@auburn.edu, or click here for the webpage.

The researchers advise that inherited macrothrombocytopenia should be suspected in any dog that has a persistently low platelet counts and that is non-responsive to treatment with antibiotics or steroids and has no history or evidence of abnormal bleeding. Since the identification of this mutation in CKCS, the identical mutation has also been documented in other breeds of dogs with inherited macrothrombocytopenia including Chihuahua, Labrador retriever, Poodle, English toy spaniel, Labradoodle, Shih Tzu, Maltese, and Jack Russell.

Editor's Note: Oversized platelets in the cavalier King Charles spaniel should not come as a surprise, and in view of the widespread extent of severely symptomatic disorders in the CKCS, such as mitral valve disease and syringomyelia, it is unreasonable to exclude cavaliers with idiopathic asymptomatic thrombocytopenia from being bred. So, we see little value in testing cavalier breeding stock to detect macrothrombocytopenia.

July 2011: Geneticist Lowell Ackerman opposes breeding cavaliers with genetic platelet disorders. In his just-released second edition of his book, Genetic Connection: A Guide to Health Problems in Purebred Dogs, geneticist Dr. Lowell Ackerman states:

"An asymptomatic thrombocytipenia typically, but not always, associated with large to giant platelets has been reported in the Cavalier King Charles spaniel. The condition, macrothrombocytopenia, has been reported as an autosomal recessive trait in the breed, and the molecular basis for the condition is a mutation in the gene encoding β-1 tubulin. The diagnosis should be suspected when thrombocytopenia and large platelets are reported in this breed without evidence of bleeding diathesis. No treatment is necessary. Despite the fact that this disorder appears to be asymptomatic, it would seem prudent not to use affected animals for breeding. Genetic testing is now available for identifying affected, carrier, and clear individuals."
Editor's Note: In view of the widespread extent of severely symptomatic disorders in the CKCS, such as mitral valve disease and syringomyelia, it is unreasonable to exclude cavaliers with idiopathic asymptomatic thrombocytopenia from being bred.

December 2010: Dr. Oyama studies links between cavaliers' giant platelets and MVD. Dr. Mark A. Oyama of the University of Pennsylvania is continuing his research into links between cavaliers' giant platelets and the breed's high incidence of mitral valve disease. His previous research has shown that CKCSs have elevated levels of serotonin (5-HT). The hypothesis is that 5-HT signaling is an important component of MVD in dogs. He suspects that the source of elevated serum 5HT is the blood platelets, since platelets are believed to contain 99% of all circulating 5-HT. It has been recently shown that platelet contents can activate disease changes within the heart muscle of experimental animals. The platelet, heart muscle, and valve-specific content of 5-HT in dogs with heart disease has not been previously reported. Dr. Oyama wants to determine if platelet 5-HT is the source of elevated serum 5-HT in cavaliers with MVD, as well as to quantify the amount of 5-HT in both the left ventricular muscle and mitral valve leaflets of affected dogs.

May 2008: Auburn University team finds gene mutation related to macrothrombocytopenia in CKCSs. Auburn University Veterinary SchoolIn a 2008 study at Auburn University (Drs. Jennifer Brooke Davis, M. Toivio-Kinnucan, S. Schuller, Mary K. Boudreaux) determined that "a mutation in the gene encoding β1-tubulin correlated with macrothrombocytopenia in CKCS." They concluded that "this information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombocytopenic CKCS with concurrent mitral valve regurgitation."

2007: Auburn Univ. student Jennifer Brooke Davis finds genetic mutation in cavaliers' blood platelets. Morris Animal Foundation reports the results of a funded study by an Auburn University student, Jennifer Brooke Davis, who examined the molecular basis for inherited macrothrombocytopenia in CKCSs.

2007: Dr. Inge Tarnow, at the University of Copenhagen's Department of Animal and Veterinary Basic Sciences, heads a group of specialists studying pathophysiological aspects of early mitral valve diseases in cavaliers, including changes in platelet function, hemostatic changes, and prognostic factors. They currently are performing a large longitudinal study of 100 Cavaliers with examinations (echocardiographic and blood tests) at ages 2, 4, and 8 years.

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Related Links

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Veterinary Resources

Macrothrombocytosis in cavalier King Charles spaniels. Brown SJ, Simpson KW, Baker S, Spagnoletti MA, Elwood CM. Vet Rec. 1994 Sep 17;135(12):281-3.  Quote: "Low platelet counts (< 150 x 10(9)/litre) have been measured by autoanalyser in cavalier King Charles spaniels with a wide range of diseases. However, the direct examination of blood smears revealed adequate numbers of platelets but many abnormally large platelets and it was not clear whether the large platelets were the result of disease or a breed abnormality. The numbers and diameters of the platelets in a group of clinically normal King Charles spaniels and a group of other breeds were measured by autoanalyser and manually. The automated platelet counts were lower in the King Charles spaniels than in the other breeds (P < 0.01), but the manual counts were similar. The platelet diameter was greater in the cavalier King Charles spaniels (median values 2.5 to 3.75 microns, P < 0.001) than the other breeds (median values 1.25 to 2.5 microns) and had a bimodal distribution with peaks at 1.25 to 2.5 microns and 3.75 to 5 microns. No clinical evidence of platelet dysfunction was detected. These findings indicate that cavalier King Charles spaniels may have large platelets which could lead to the erroneous diagnosis of thrombocytopenia by automated haematological analysis."

Thrombocytopenia in the Cavalier King Charles Spaniel. Eskell P, Häggström J, Kvart C, Karlsson A. J Small Anim Pract 1994; 35:153-155.

Idiopathic, asymptomatic thrombocytopenia in Cavalier King Charles spaniels: 11 cases (1983-1993).  Smedile LE, Houston DM, Taylor SM, Post K, Searcy GP. J Am Anim Hosp Assoc. 1997 Sep-Oct;33(5):411-5.

Macrothrombocytosis in a Cavalier King Charles Spaniel. Kraus MS. Veterinary Forum, May, 1997.

Update on Mitral Valve Disease. Jens Häggström and Clarence Kvart. Proc. 15th ACVIM Forum; 1997. Quote: "An interesting observation that may be of comparative interest is that Cavalier King Charles Spaniels have been shown to have a high prevalence (30%) of thrombocytopenia and macrothrombocytosis. Humans with MVP [mitral valve prolapse] tend to have shortened platelet survival times and thromboembolic episodes primarily in the retinal and cerebral circulation. Thromboembolic events in the retinal ore cerebral circulation may be involved in the disturbances described in the breed as 'episodic falling' and 'fly catching'."

Platelet Counts in Cavalier King Charles Spaniels. Antech Diagnostics News, July 1998.

Control of Canine Genetic Diseases. Padgett, G. A., Howell Book House 1998, pp. 198-199, 223.

Increased platelet aggregation response in Cavalier King Charles spaniels with mitral valve prolapse. Olsen,L.H., Kristensen, A. T., Häggström, J., Jensen, A. L., Klitgaard, B., Hansson, H., Pedersen, H. D. J.Vet.Int.Med. 2001;15:209-216. Quote: "In Cavalier King Charles Spaniels (CKCS), a breed predisposed to myxomatous mitral valve disease, there is a high prevalence of hypomagnesemia and platelet anomalies, such as thrombocytopenia and macrothrombocytosis. The objective of this study was to evaluate platelet aggregation responses in CKCS and to determine the relationship between the platelet aggregation response and serum magnesium concentration, MVP, mitral regurgitation (MR), and platelet count. ... In conclusion, CKCS with MVP appeared to separate into 2 groups—1 group with <100,000 platelets/μL, normal platelet aggregation, low serum magnesium concentration, and enlarged platelets, and another group with >100,000 platelets/μL, increased platelet aggregation, and normal serum magnesium concentration and platelet size."

Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles Spaniels is an autosomal recessive trait. Pedersen HD, Häggström J, Olsen LH, Christensen K, Selin A, Burmeister ML, Larsen H. J Vet Intern Med 2002;16(2):169-73. Quote: "Cavalier King Charles Spaniels (CKCS) often have idiopathic asymptomatic thrombocytopenia. In affected dogs, the thrombocytes often are large, and it has been speculated that the condition could be an inherited macrothrombocytopenia. The aim of this study was to examine the inheritance of idiopathic, asymptomatic thrombocytopenia in CKCS. Sixteen families (both parents and ≥3 offspring) of privately owned CKCS were included. There were 105 clinically healthy dogs (50 from Denmark and 55 from Sweden): 81 offspring and 26 parents (2 dogs had both roles). Because autoanalyzers have difficulty counting large platelets, the platelets were counted manually, with a counting chamber. Platelet counts were not influenced by age, gender, or heart murmur status. Thrombocytopenia (≤100,000 platelets/|μL) was found in 46% of the parents. The pedigrees indicated that thrombocytopenia segregated as an autosomal recessive trait and that 100,000 platelets/|μL was appropriate as a lower limit of normal. Affected offspring were found in all families, showing that all of the included parents were at least carriers. Therefore, the expected segregation ratios (which were in good accordance with the observed ones) were 1:0, 1:1, and 1:3 for the 3 crosses: affected X affected, normal X affected, and normal X normal. Within a given cross, the mean parental platelet count had no influence on the platelet counts of the offspring. We conclude that idiopathic, asymptomatic thrombocytopenia in CKCS is inherited in an autosomal recessive manner. The condition most likely constitutes an inherited macrothrombocytopenia in dogs."

Intact Primary Hemostatic Responses in Cavalier King Charles Spaniels with Macrothrombocytopenia. AT Kristensen, H Texel, HD Pedersen. J Vet Intern Med 2002; 16:348. Quote: “Cavalier King Charles Spaniels (CKCS) have a high prevalence of inherited macrothrombocytopenia with no apparent clinical signs of bleeding. In a study using platelet rich plasma and aggregometry thrombocytopenic CKCS did not have increased platelet responses. Recently a more physiologic method, the PFA-100 (Dade-Behring), has become available for evaluation of primary hemostatic function. ...This shows that primary hemostatic responses of CKCS are intact despite severe thrombocytopenia.”

Quantitative, functional, and morphologic characterization of platelets in the Cavalier King Charles spaniel. Cowan SM, Carroll RC, Gerard D, Bartges JW, et al. J Vet Intern Med 2002;16:349.  Quote: "Thrombocytopenia and macrothrombocytes have been reported to occur in Cavalier King Charles Spaniels (CKCS), but the clinical characteristics of a thrombocytopathy in CKCS are unknown. The purposes of this study were to describe quantitative, functional, and morphological characteristics of platelets in the CKCS. Blood (25 ml) from 42 clinically normal CKCS was collected from the jugular vein and analyzed within 1 hour of collection. Automated platelet counts were obtained using citrate and EDTA as anticoagulants. Manual platelet counts were performed on EDTA blood in hemacytometer chambers, in duplicate, using phase contrast microscopy. Percent platelet aggregation in response to increasing concentrations of ADP (2, 4, 8, 16, 32 uM) was determined on platelet-rich plasma prepared from citrated blood. Standard, whole mount, and scanning electron microscopy (EM) was performed to examine internal morphology, distribution of dense granules, and platelet size. A cardiologist (REG) recorded the grade, character and site of murmurs. Thrombocytopenia ( 150,000/ul) using manual platelet counts was present in 29/42 dogs (69%). Average manual platelet count was 119,817/ul. One dog had platelet clumping and was excluded from analysis. There was a good correlation (r2 83%, p .001) between manual and automated platelet counts using EDTA, and a good correlation (r2 82, p .001) between manual and automated platelet counts using citrate. There were no significant differences in factors causing variation in platelet counts (specimen collection or handling, methodology, mean RBC volume, or PCV) in dogs that were thrombocytopenic. Twenty of forty-two dogs (48%) had cardiac murmurs. There was no correlation between thrombocytopenia and murmurs, sex, age, or hair coat color. There was no significant difference in percent aggregation in response to varying concentrations of ADP in CKCS with thrombocytopenia. There was also no significant difference in percent aggregation in CKCS with murmurs compared with those without murmurs. We conclude that EDTA or citrate can be used as anticoagulants for automated platelet counts in CKCS. There was no association of thrombocytopenia with murmurs, and no association of abnormal platelet aggregation with murmurs or thrombocytopenia. Further studies are underway to identify structural abnormalities in platelet morphology, granule content, or size in CKCS with thrombocytopenia."

Duncan & Prasse’s Veterinary Laboratory Medicine: Clinical Pathology. 4th ed. Latimer KS, Mahaffey EA, Prasse KW (eds). Iowa State Press, 2003, pp. 101-102, 114-116.

Decreased Platelet Function in Cavalier King Charles Spaniels with Mitral Valve Regurgitation. Tarnow I., Kristensen A. T., Texel H., Olsen L. H., Pedersen H. D.; Vol. 17, No. 5, pp. 680–686. J Vet Intern Med. 2003;17(5):680-686. Quote: "With aggregometry, increased platelet activity has been reported in Cavalier King Charles Spaniels (CKCS) without mitral regurgitation (MR). In contrast, dogs with MR have been found to have decreased platelet activity. The purpose of this study was to test an easy bedside test of platelet function (the Platelet Function Analyzer [PFA-100®]) to see if it could detect an increase in platelet activity in CKCS without MR and a decrease in platelet activity in CKCS with MR. ... CKCS with mild, moderate, and severe, clinically inapparent MR have decreased platelet function. The previous finding of increased platelet reactivity in nonthrombocytopenic CKCS without MR could not be reproduced with the PFA-100 device."

Platelet Counts in Cavalier King Charles Spaniels. Antech Diagnostics News Lab Tips, Oct 2003.

Guide to Congenital and Heritable Disorders in Dogs. Dodds WJ, Hall S, Inks K, A.V.A.R., Jan 2004, Section II(311a).

Breed Predispositions to Disease in Dogs & Cats. Alex Gough, Alison Thomas. 2004; Blackwell Publ. 44-45.

Comparison of manual and automated methods for determining platelet counts in dogs with macrothrombocytopenia. Olsen LH, Kristensen AT, Qvortrup K, Pedersen HD; J Vet Diagn Invest, March 2004; 16:167-170. Quote: "Platelet counts were performed in 43 Cavalier King Charles Spaniels (CKCS), a breed predisposed to macrothrombocytopenia) and in 10 control dogs using 3 automated systems and 3 manual methods (erythrocyte-lysing agents + counting chamber or evaluation of blood smear). Good correlations were found between platelet counts using all methods (all P < 0.0001; R2 = 0.71-0.85). Best correlations were found between the manual methods. Significantly larger platelets were found in CKCS with platelet count < or = 100,000/microl when compared with control dogs and CKCS with platelet count > 100,000/microl (both P < 0.0001). All platelet counts--except when made with the 2 counting chamber methods--were underestimated at platelet counts < or = 100,000/microl."

Giant platelet disorder in the Cavalier King Charles Spaniel. Cowan SM, Bartges JW, Gompf RE, et al.; Exp Hem, April 2004; 32:344-350. Quote: "A benign inherited giant platelet disorder affects approximately 50% of Cavalier King Charles Spaniels. It is characterized by thrombocytopenia, macrothrombocytes, or decreased platelet aggregation in response to ADP. Platelet ultrastructure is normal. Citrated or EDTA blood provides accurate platelet counts. Further studies are indicated to determine platelet glycoprotein structure and any association with mitral endocardiosis. Cavaliers may be useful models of inherited giant platelet disorders."

Assessment of changes in hemostatic markers in Cavalier King Charles Spaniels with myxomatous mitral valve disease. Tarnow I, Kristensen AT, Olsen LH, Pedersen HD. Am J Vet Res. 2004 Dec;65(12):1644-52. Quote: "Objective - To evaluate markers of haemostasis and their relationship to the degree of mitral regurgitation (MR) and platelet function in Cavalier King Charles Spaniels (CKCSs) with myxomatous mitral valve disease. ... Conclusions and Clinical Relevance - In CKCSs, MR appeared to be associated with a low plasma vWF concentration and likely a loss of vWF HMWMs (possibly through their destruction via shear stress to the blood). The importance of the changes in plasma fibrinogen concentration and the thromboembolic risk in dogs with MR remain to be investigated."

Biomarkers of Platelet Activation in Cavalier King Charles Spaniels. D.F. Hogan, D.J. Weiss, C.A. Thompson, M.P. Ward. J Vet Intern Med; May/June 2005;19(3) (ACVIM 23rd Ann. Vet. Med. Forum Abstract Program: Abstract 175).

Platelets in Cavalier King Charles Spaniels. Antech Diagnostics News Lab Tips, June 2005.

Dogs with Heart Diseases Causing Turbulent High-Velocity Blood Flow Have Changes in Platelet Function and von Willebrand Factor Multimer Distribution. Tarnow I., Kristensen A. T., Olsen L. H., Falk T., Haubro L., Pedersen L. G., Pedersen H. G.; J Vet Intern Med. 2005 Jul;19(4):515-521. Quote: "The purpose of this prospective study was to investigate platelet function using in vitro tests based on both high and low shear rates and von Willebrand factor (vWf) multimeric composition in dogs with cardiac disease and turbulent high-velocity blood flow. Client-owned asymptomatic, untreated dogs were divided into 4 groups: 14 Cavalier King Charles Spaniels (Cavaliers) with mitral valve prolapse (MVP) and no or minimal mitral regurgitation (MR), 17 Cavaliers with MVP and moderate to severe MR, 14 control dogs, and 10 dogs with subaortic stenosis (SAS). Clinical examinations and echocardiography were performed in all dogs. PFA100 closure times (the ability of platelets to occlude a hole in a membrane at high shear rates), platelet activation markers (plasma thromboxane B2 concentration, platelet surface P-selectin expression), platelet aggregation (in whole blood and platelet-rich plasma with 3 different agonists), and vWf multimers were analyzed. Cavaliers with moderate to severe MR and dogs with SAS had longer closure times and a lower percentage of the largest vWf multimers than did controls. Maximal aggregation responses were unchanged in dogs with SAS but enhanced in Cavaliers with MVP (regardless of MR status) compared with control dogs. No significant difference in platelet activation markers was found among groups. The data suggest that a form of platelet dysfunction detected at high shear rates was present in dogs with MR and SAS, possibly associated with a qualitative vWf defect. Aggregation results suggest increased platelet reactivity in Cavaliers, but the platelets did not appear to circulate in a preactivated state in either disease."

Idiopathic thrombocytopenia in Cavalier King Charles Spaniels. Singh M. K., Lamb W. A. Aust Vet J. 2005 Nov;83(11):700-3.  Quote: "It has been shown that Cavalier King Charles Spaniels (CKCS) in Sweden and Denmark have a high prevalence of idiopathic thrombocytopaenia without bleeding tendencies and that the syndrome has an autosomal recessive inheritance pattern. A similar thrombocytopaenia has been observed in New South Wales (NSW). The purpose of this study was to determine the prevalence of asymptomatic idiopathic macrothrombocytopaenia in the population of CKCS in NSW and to determine if it exhibits an autosomal recessive inheritance pattern. We also aimed to determine if significant differences existed when counting platelets manually, by auto analyser or by blood smear estimation in CKCS and mixed breed dogs. Blood was collected from 172 dogs (152 CKCS and 20 mixed breed) and placed into sodium-citrate anticoagulant. Platelet counts were performed manually, by auto analyser and by blood smear estimates in CKCS and mixed breed dogs. Blood smears were also examined for platelet clumping and morphology as well as erythrocyte and leukocyte morphology. Pedigree analysis was performed to determine if an autosomal recessive inheritance pattern was supported. A statistically significant difference was found in platelet counts between CKCS and mixed breed dogs (P < 0.0001). CKCS had a platelet count that was 32% that of the controls (95% confidence interval = 28 to 37%). If the lower limit of normal was set at 200 x 109/L, the prevalence of thrombocytopaenia in CKCS was 90% with 25% having severe thrombocytopaenia (<50 x 109/L). There was no significant difference between methods used to count platelets. Thirty percent of CKCS had macrothrombocytes. Pedigree analysis and examination of obtained and expected segregation ratios from 17 CKCS families supported an autosomal recessive pattern of Mendelian inheritance."

Macrothrombocytopenia in the Cavalier King Charles Spaniel. Meg Carriere, DVM; Kenneth S. Latimer, DVM, PhD; Bruce E. LeRoy, DVM, PhD; Heather L. Tarpley, DVM. College of Veterinary Medicine, Univ. of Georgia. 2006. www.vet.uga.edu/vpp/clerk/carriere/index.php

Platelet function in dogs: breed differences and effect of acetylsalicylic acid administration. Nielsen LA, Zois NE, Pedersen HD, Olsen LH, Tarnow I. Vet Clin Pathol. 2007 Sep;36(3):267-73. Quote: "The objective of this study was to investigate platelet function in clinically healthy dogs of 4 different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], ...) were included in the study. ... A higher platelet aggregation response was found in CKCS compared to the other breeds. ... Platelet function is influenced by breed, depending upon the methodology applied. However, the importance of these breed differences remains to be investigated."

Comparison of methods for determining platelet numbers and volume in cavalier King Charles spaniels. W. Bertazzolo, S. Comazzi, L. Sesso, P. Scarpa, G. Ru, S. Paltrinieri. J Small Animal Practice, Oct 2007;48(10):556-561. Quote: "CKCS with macrothrombocytosis have low platelet counts on impedance cell counters, laser cell counters and microscopic estimation. CKCS with low platelet counts may have a normal platelet crit detected by a quantitative buffy coat analyser and thus a normal circulating platelet mass."

The molecular basis for inherited macrothrombocytopenia in Cavalier King Charles Spaniels: Genetic Insight into Blood Disorder. Jennifer Brooke Davis. Morris Animal Foundation Study ID: D06CA-611. Quote: "The focus of this study was the role of a protein found in healthy, mature platelets. Through analysis of this protein, the researchers were able to identify a genetic mutation in the protein of dogs affected. This finding could help researchers design a more precise test for certain platelet disorders, ultimately eliminating them through responsible breeding. The ability to distinguish acquired from genetic forms of diseases also will help researchers to more precisely design their treatments and avoid use of unnecessary steroids, antibiotics or other medications."

Mutation in β1-Tubulin Correlates with Macrothrombocytopenia in Cavalier King Charles Spaniels. B. Davis, M. Toivio-Kinnucan, S. Schuller, M.K. Boudreaux. J.Vet.Int.Med. May-June 2008;22(3): 540-545. Quote: "Background: Cavalier King Charles Spaniels (CKCS) have a high prevalence of inherited macrothrombocytopenia. The purpose of this study was to determine if a mutation in β1-tubulin correlated with presumptive inherited macrothrombocytopenia. Hypothesis: A mutation in β1-tubulin results in synthesis of an altered β1-tubulin monomer. α-β tubulin dimers within microtubule protofilaments are unstable, resulting in altered megakaryocyte proplatelet formation. ... Methods: DNA was used in polymerase chain reaction (PCR) assays to evaluate β1-tubulin. Platelet numbers and mean platelet volume (MPV) were evaluated for a correlation with the presence or absence of a mutation identified in β1-tubulin. Platelets obtained from homozygous, heterozygous, and clear CKCS were further evaluated using electron microscopy and immunofluorescence. Results: A mutation in the gene encoding β1-tubulin correlated with macrothrombocytopenia in CKCS. Electron microscopy and immunofluorescence studies suggest that platelet microtubules are present but most likely are unstable and decreased in number. Conclusions and Clinical Importance: The macrothrombocytopenia of CKCS correlated with a mutation in β1-tubulin. α–β tubulin dimers within protofilaments most likely are unstable, leading to altered proplatelet formation by megakaryocytes. This information will aid in distinguishing inherited from acquired thrombocytopenia. It also provides insight into the mechanism of platelet production by megakaryocytes, and also may prove useful in understanding heart-related changes in macrothrombo-cytopenic CKCS with concurrent mitral valve regurgitation."

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels. Harold Tvedten, Inger Lilliehöök, Anna Hillström, Jens Häggström. Vet. Clinical Pathology; July 2008; 37(3): 266-271. Quote: "Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKCS than methods directly enumerating platelet numbers. Objectives: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs. ... The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). ... The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs. ... Results: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 × 109 platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum. Conclusions: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass."

Platelet distribution width and mean platelet volume in the interpretation of thrombocytopenia in dogs. N. X. Bommer, D. J. Shaw, E. M. Milne, A. E. Ridyard. J.Sm.An.Prac.;Oct 2008;49(10):518-524. Quote: "Objectives: To investigate the use of platelet volume indices in the interpretation of thrombocytopenia in dogs with systemic disease. Methods: Case records of 80 control dogs and 159 thrombocytopenic dogs with systemic disease were reviewed retrospectively. ... Cavalier King Charles spaniels were excluded from all groups because of known breed- related macrothrombocytopenia. ... The relationships between mean platelet volume, platelet distribution width and platelet count in systemically well dogs were established. Knowledge of these relationships was used to interpret thrombocytopenia in terms of the underlying disease process. Results: There was a positive relationship between platelet distribution width and mean platelet volume. Both platelet distribution width and mean platelet volume varied in a negative relationship with platelet count, which was unaffected by signalment, underlying disease, variations in haematological parameters or serum proteins. Clinical Significance: The interpretation of mean platelet volume and platelet distribution width in dogs requires simultaneous reference to the platelet count. Platelet volume indices did not aid interpretation of thrombocytopenia in terms of underlying pathological processes in the population studied."

Updates in clinical pathology: Macrothrombocytopaenia in Cavalier King Charles Spaniels. Size matters! Sarah Putwain. Companion Animal Oct 2009 14(8):36-38.

Insights into Serotonin Signaling Mechanisms Associated with Canine Degenerative Mitral Valve Disease. M.A. Oyama and R.J. Levy. J Vet Intern Med Jan/Feb 2010;24:27–36. Quote: "In Cavalier King Charles Spaniels, the time from onset of a heart murmur to death because of congestive heart failure can be quite accelerated, highlighting the rapid nature of disease progression in this particular breed. ... Putative sources of heightened 5HT (serotonin) signaling in dogs with DMVD include increased circulating or platelet-derived 5HT,increased autocrine 5HT production, or valvular SERT deficiency. We have previously reported increased serum 5HT concentrations in dogs with DMVD (degenerative mitral valve disease). Serum 5HT was increased by approximately 50% in dogs with DMVD as well as in small breed dogs predisposed to DMVD as compared with healthy large breed control dogs. Interestingly, Cavalier King Charles Spaniels, which are highly predisposed to DMVD as well as macrothrombocytosis, had significantly higher serum 5HT concentrations than did other breeds of dogs. In general, measurement of serum 5HT encompasses 2 different sources, platelet-stored 5HT and plasma 5HT, of which platelet 5HT is the much larger source. The hypothesis that platelet-derived 5HT directly activates myocardial or valvular fibroblasts is supported by Yabanoglu et al who reported chemotaxis of platelets to regions of cardiac damage, with subsequent 5HT release, fibroblast differentiation, and production of TGF-b1 and matrix metalloproteinases. This effect was inhibited by co-administration of ketanserin, a 5HT–R2Aspecific blocking agent, again suggesting that this receptor plays an important role in the activation of cardiac fibroblasts by 5HT. This study is important insofar as it supports a key hypothesis involving direct interaction among platelets, platelet-derived 5HT, 5HT receptors, increased 5HT and TGF-β1 signaling, and subsequent cardiac cell differentiation."

Thromboelastography in Dogs with Asymptomaticmyxomatous Mitral Valve Disease. I Tarnow, LH Olsen, SG Moesgaard, AU Martinsen, AC Birkegaard, AT Kristensen, B Wiinberg. J Vet Intern Med 2010;24:--- (ACVIM 28th Ann. Vet. Med. Forum Abstract Program: Abstract 11). Quote: "Changes in platelet aggregation responses and von Willebrand factor multimer levels have been reported in dogs with asymptomatic heart disease. Alterations in hemostatic pathways may be involved in the progression of canine mitral valve disease by causing microthrombosis and vessel changes in the myocardium, and it is being debated whether dogs with heart disease could benefit from antithrombotic therapy. We hypothesized that a global hypercoagulability is present in dogs with asymptomatic mitral valve disease. The study investigated Kaolin and Tissue Factor activated thromboelastographic (TEG) tracings in 3 groups of dogs: 11 Cavalier King Charles Spaniels (CKCS) with no or minimal mitral regurgitation (MR) (age 5.0 1.8 years), 14 CKCS with severe MR (regurgitant jet size by echocardio-graphy 50%) (age 7.1 1.6 years), and 8 healthy Beagles with no or minimal MR (age 7.7 2.3 years). ... These data do not support the hypothesis that a global hypercoagulability is present in CKCS with asymptomatic myxomatous mitral valve disease. Moreover, prospective longitudinal trials are needed to demonstrate a benefit of anti-thrombotic therapy in dogs with asymptomatic heart disease. The data supports previous findings that CKCS with macrothrombocytopenia have a normal coagulation response despite low platelet counts."

Breed Predispositions to Disease in Dogs & Cats (2d Ed.). Alex Gough, Alison Thomas. 2010; Wiley-Blackwell Publ. 54.

Genetic Connection: A Guide to Health Problems in Purebred Dogs, Second Edition. Lowell Ackerman. July 2011; AAHA Press; pg 88. Quote: "An asymptomatic thrombocytipenia typically, but not always, associated with large to giant platelets has been reported in the Cavalier King Charles spaniel. The condition, macrothrombocytopenia, has been reported as an autosomal recessive trait in the breed, and the molecular basis for the condition is a mutation in the gene encoding β-1 tubulin. The diagnosis should be suspected when thrombocytopenia and large platelets are reported in this breed without evidence of bleeding diathesis. No treatment is necessary. Despite the fact that this disorder appears to be asymptomatic, it would seem prudent not to use affected animals for breeding. Genetic testing is now available for identifying affected, carrier, and clear individuals."

Inherited platelet disorders. Mary K. Boudreaux. J.Vet.Emer. & Crit. Care. Jan.2012.

Validation of Advia plateletcrit for assessing platelet mass in dogs, including Cavalier King Charles Spaniels. Harold W. Tvedten, Inger E. Lilliehöök, Josefine Öberg, Jens Häggström, Katja Höglund and Ingrid Ljungvall. Vety. Clinical Pathology.Sept 2012;41(3):336-343. Quote: "Background: Determination of the plateletcrit (PCT) is the most effective way to evaluate platelet mass in dogs, such as Cavalier King Charles spaniel (CKCS) dogs, with macrothrombocytopenia. The IDEXX VetAutoread hematology analyzer, which performs quantitative buffy coat (QBC) analysis, has been validated to determine platelet mass in CKCS dogs. The Advia 2120 reports a PCT, but the validity of this value has not been evaluated for dogs with macrothrombocytopenia. Objectives: The goal of this study was to validate MPV and PCT determined by the Advia 2120 in dogs, including CKCS dogs, comparing values with those obtained from QBC analysis. Methods: Advia PCT was compared with QBC results from 43 CKCS dogs and 15 dogs of other breeds in one study. Advia PCT, platelet count, and MPV were evaluated to identify biologic patterns in 31 clinically healthy CKCS dogs and 66 dogs of 3 other breeds and to generate values used for comparisons. Results: Advia PCT agreed well with QBC results in general, but had a negative bias and appeared to underestimate PCT in CKCS dogs with the lowest PCTs. Advia PCT and MPV results followed expected biologic patterns in CKCS dogs and dogs of other breeds with MPVs being highest in dogs with the lowest platelet counts. Conclusions: Advia 2120 PCT and MPV satisfactorily identified changes in platelet mass and size in CKCS dogs, but PCTs were lower than expected, especially in CKCS dogs with the lowest PCTs, when compared with QBC results."

Macrothrombocytopenia in a Cavalier King Charles Spaniel. Sarah Schmidt & Joyce S. Knoll. Clinician's Brief. Dec. 2012;10(12):31-34. Quote: "History: The Cavalier King Charles spaniel (CKCS) had a previous diagnosis of a grade III/VI left systolic heart murmur. Physical Examination: The heart murmur as previously described was detected. Instability in the left stifle was also noted. The remainder of the examination was unremarkable. Laboratory Evaluation: CBC was unremarkable with the exception of an automated platelet count of 2350/μL (reference interval, 181–525 ¥ 103/μL; Cell-Dyn 3500 hematology analyzer [abbott.com] using impedance technology for platelet count). A blood smear was evaluated. Diagnosis: ... It is important to note that these dogs may mistakenly be considered to have an underlying disease that results in thrombocytopenia, such as immune-mediated thrombocytopenia, tick-borne infection, or a bone marrow disorder. Unlike other dogs with platelet counts of 30,000/μL or lower, CKCSs with the mutation do not have signs of spontaneous mucocutaneous hemorrhage because their overall platelet function is normal. Platelet function is believed to depend more on total platelet mass (number × volume) than platelet number alone. The platelet mass of CKCSs with inherited macrothrombocytopenia is reportedly similar to CKCSs without the mutation (normal platelet count and no large platelets). ... Follow-up: As the dog did not have signs to suggest an underlying bleeding disorder or a hyper coagulative state, thrombocytopenia and macroplatelets were presumed to be a result of inherited macrothrombocytopenia. The dog was also diagnosed with a cranial cruciate tear of the left stifle."

Platelet count and plateletcrit in Cavalier King Charles Spaniels and Greyhounds using the Advia 120/2120. Jessica Anne Kelley. Univ. of Minn. Digital Conservancy. Jan. 2013. MS thesis. Quote: "This thesis investigates the use of the plateletcrit as an assessment of total platelet biomass in Cavalier King Charles Spaniels with asymptomatic congenital macrothrombocytopenia. ... Background: Models for the regulation of thrombopoiesis predict that platelet mass is the biologically regulated parameter, yet clinical evaluations are often based on platelet number. When there is concurrent thrombocytopenia and variation in platelet size, platelet count may poorly estimate total platelet mass, confounding clinical decision making. Objective: We hypothesize that plateletcrit provides clinically important information when platelet number is an incomplete representation of platelet mass, such as in genetic macrothrombocytopenia. Methods: We retrospectively compared platelet count and plateletcrits with general reference intervals for four groups of dogs: sick Cavalier King Charles Spaniels and Greyhounds that presented for non-hematopoietic disease to the University of Minnesota (Advia 2120) and Auburn University Teaching Hospitals (Advia 120) over a 3 year period. Results: A canine plateletcrit reference interval of 0.129 – 0.403 % was established. None of the four sample groups were found to have significantly more individuals below the reference interval for plateletcit. For platelet count, only the Cavalier groups were found to have significantly more individuals below the reference interval than predicted. Conclusion: Use of the plateletcrit as determined by the Advia 120/2120 appeared to avoid overestimation of low platelet mass in sick Cavalier Kind Charles Spaniels in a clinical setting. In contrast, the plateletcrit performed similarly to the platelet count in evaluation of platelet mass in sick Greyhounds. ... In this particular model of an inherited asymptomatic macrothrombocytopenic platelet disorder, Cavalier King Charles Spaniels represent a group of individuals in which the bleeding risk is better evaluated using plateletcrit than platelet number. The platelet mass appears significantly lower in affected dogs when measured by count, but when measured by plateletcrit, more of the patients are appropriately within the reference range. Therefore, for the Cavalier King Charles Spaniels affected by this mutation, plateletcrit better represents the true total platelet biomass in peripheral circulation." (See also, published version of this article, below.)

In vitro effect of pimobendan on platelet aggregation in dogs. Eryn A. Shipley, Daniel F. Hogan, Nonya N. Fiakpui, Aliya N. Magee, Henry W. Green III, Kimberly A. Sederquist. Amer,J,Vety.Res. March 2013. 74(3):403-407. Quote: "Objective: To determine whether pimobendan has in vitro antithrombotic properties through inhibition of platelets in canine blood samples. Animals: 10 healthy adult dogs.  ... Results: Compared with findings for 0.0μM pimobendan, composite platelet aggregation (area under the curve [AUC]) and maximal platelet aggregation (aggregation units [AUs]) at 10.0μM pimobendan were significantly decreased for collagen-induced aggregation (AUC, 349.7 ± 58.4 vs 285.1 ± 72.2; maximal platelet aggregation, 196.2 ± 25.8 AUs vs 161.5 ± 38.0 AUs), and the AUC and velocity of aggregation at 10.0μM pimobendan were significantly decreased for ADP-induced aggregation (AUC, 268.5 ± 35.1 vs 213.4 ± 77.2; velocity of aggregation, 15.7 ± 2.9 AUs/min vs 11.8 ± 3.5 AUs/min). Pimobendan had no significant effect on plasma thromboxane concentration or thromboelastographic variables, regardless of concentration. Conclusions and Clinical Relevance: In vitro, pimobendan had an antiplatelet effect in canine blood samples at a concentration 1,000-fold higher than that clinically achievable. These antiplatelet properties do not appear to contribute to the positive clinical profile of the drug in dogs. Pimobendan administration would not appear to confer a risk for bleeding and does not have to be avoided in dogs with thrombocytopenia or those concurrently receiving antiplatelet drugs."

Platelet count and plateletcrit in Cavalier King Charles Spaniels and Greyhounds using the Advia 120 and 2120. Jessica Kelley, Leslie C. Sharkey, Pete W. Christopherson, Aaron Rendahl. Vet. Clinical Pathology. March 2014;43(1):43-49. Quote: "Background: Models for the regulation of thrombopoiesis predict that platelet mass is the biologically regulated variable, yet clinical evaluations of thrombopoiesis are often based on platelet number. When thrombocytopenia and variation in platelet size occur concurrently, platelet count may poorly estimate total platelet mass, confounding clinical decision making. Objective: We hypothesized that plateletcrit (PCT) provides clinically important information when platelet number is an incomplete representation of platelet mass, such as in genetic macrothrombocytopenia. Methods: We retrospectively compared platelet count and PCTs with general reference intervals for 4 groups of dogs: sick Cavalier King Charles Spaniels (CKCS) and Greyhounds, presented for nonhematopoietic disease to the University of Minnesota (measured using an Advia 2120) and Auburn University Teaching Hospitals (measured using an Advia 120) over a 3-year period. Results: A canine PCT reference interval of 0.129–0.403% was established. None of the 4 sample groups had significantly more individuals below the reference interval for plateletcrit. For platelet count, only the 2 CKCS groups had significantly more individuals below the reference interval than predicted. Conclusion: Use of the PCT as determined by the Advia 120/2120 appeared to avoid overestimation of low platelet mass in sick CKCS in a clinical setting. In contrast, the PCT performed similarly to the platelet count in evaluation of platelet mass in sick Greyhounds. Evaluation of the PCT should be considered in other conditions associated with increased mean platelet volume." (See also, Ms. Kelley's MS thesis, above.)

High-level serotonin-binding in subpopulation of highly-activated platelets in cavalier King Charles spaniels with myxomatous mitral valve disease. S.E. Cremer, A.T. Kristensen, M.J. Reimann, N.B. Eriksen, S.F. Petersen, C.B. Marschner, I. Tarnow, M.A. Oyama, L.H. Olsen. J.Vet.Int.Med. Apr. 2014;28(3). Quote: "Circulating (platelet-derived) serotonin (5HT) concentration is elevated in early stages of myxomatous mitral valve disease (MMVD) in Cavalier King Charles Spaniels (CKCS). CKCS are predisposed to early-onset MMVD and approximately 33% exhibit non-clinical macrothrombocytopenia (TCP). By means of flow cytometry, we investigated percent (%) and level (mean-fluorescence-intensity/MFI) of platelet-activation (CD62 surface expression) and surface-bound 5HT in platelet rich plasma (PRP) in CKCS with MMVD. Dogs with TCP were included, but only normal-size platelets were analyzed. Effect of disease-group, age, gender, body weight, hematocrit and TCP were included in multiple linear regression analyses. Disease-groups (assessed echocardiographically) were: healthy (n = 14), mild MMVD (n = 18), moderate-severe MMVD (n = 19) and severe MMVD in treatment for heart-failure (n = 10). There was an overall difference in level of platelet-activation (P = 0.04) with a tendency to higher activation level in moderate-severe MMVD (1661MFI (1356-2379)) (median (interquartile range)) and heart-failure (1452MFI (989-3242)) compared to healthy dogs (1325MFI (904-2505) (both P = 0.07)). In 28 dogs, a subpopulation of platelets with high 5HT-binding (5HT-P) was identified. These dogs (compared to non-5HT-P dogs) had higher percent of 5HT-positive platelets (10.4% (7.8-15.4) versus 5.7% (3.3-8.6), P < 0.0001), 5HT-binding level (30360MFI (26400-39260) versus 1230MFI (810-2830), P < 0.0001) and platelet-activation level (2360MFI (1520-2830) versus 1170MFI (890-1360), (P = 0.002). 5HT-P were present in 93.8% of dogs with TCP and in 29.6% of non-TCP dogs. In conclusion, a potential association between level of platelet activation and MMVD-severity was found. Interestingly, a highly-activated platelet subpopulation with high-level 5HT-binding was identified and strongly associated with TCP. Further investigation into significance of 5HT-P in CKCS is warranted."

Serotonin concentration is elevated in platelet rich plasma and left ventricular myocardial and mitral valve leaflet tissue in dogs with myxomatous mitral valve disease. S.E. Cremer, G.E. Singletary, L.H. Olsen, K. Wallace, J. Häggström, I. Ljungvall, K. Höglund, C.A. Reynolds, N. Pizzinat, M.A. Oyama. J.Vet.Int.Med. July 2014;28(4):1351. Quote: "Heightened serotonin (5HT) signaling is postulated in the development and/or progression of myxomatous mitral valve disease (MMVD) in dogs. Little is known regarding platelet, myocardial, or valvular 5HT concentration ([5HT]) in affected dogs. We sought to quantify [5HT] in platelet-rich (PRP), platelet-poor plasma (PPP), left ventricular myocardium (LV), and mitral valve leaflet tissue (MV) using HPLC. PPP/PRP was prepared from 45 dogs comprising 4 different plasma groups (pG1, healthy non-Cavalier King Charles Spaniels [CKCS], n = 8; pG2, healthy CKCS, n = 12; pG3, MMVD CKCS, n = 14; pG4, MMVD non-CKCS, n = 11). PRP [5HT] was greater than PPP [5HT] (P < 0.0001) indicating platelets as the primary source of circulating 5HT. Median PRP platelet [5HT] was significantly different between groups (P = 0.003). PRP [5HT] was greater in pG2 (1.83 femtograms/platelet[range,0.2-4.76];P = 0.002), pG3 (1.58,0.70-4.03;P = 0.005), and pG4 (1.72,0.85-4.44;P = 0.003) vs. pG1 (0.915,0.63-1.3). There was no significant difference in median PPP [5HT] between groups. At euthanasia, LV and MV tissue was obtained from 24 dogs comprising 3 different tissue groups (tG1, MMVD, n = 8; tG2, dilated cardiomyopathy [DCM]/arrhythmogenic right ventricular cardiomyopathy [ARVC], n = 7; tG3, extra-cardiac disease, n = 9). Median LV [5HT] was significantly different (P = 0.0024) between groups. LV [5HT] was higher in tG1 (11.8 ng/mg,3.96-104.83) vs. tG2 (0.87,0-10.1;P = 0.011) and vs. tG3 (2.46,0-6.94;P = 0.001). MV [5HT] was significantly different between groups (P = 0.033) namely between tG1(32.4 ng/mg,8.36-106.73) vs. tG3(3.59,0-28.26;P = 0.01). In conclusion, platelet [5HT] was elevated in healthy and MMVD CKCS compared to healthy non-CKCS. LV [5HT] was elevated in MMVD compared to DCM/ARVC or non-cardiac disease. MV [5HT] was elevated in MMVD compared to non-cardiac disease."

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