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700 Australian Veterinary Journal Volume 83, No 11, November 2005

Idiopathic thrombocytopenia in Cavalier King CharlesSpaniels

MK SINGH and WA LAMBVeterinary Specialist Centre, PO Box 307, North Ryde, New South Wales, 2113

Objective To determine the prevalence of asymptomaticidiopathic macrothrombocytopenia in the population of CavalierKing Charles Spaniels (CKCS) in New South Wales (NSW) andto determine if it exhibits an autosomal recessive inheritancepattern. We also aimed to determine if significant differencesexisted when counting platelets manually, by auto analyser orby blood smear estimation in CKCS and mixed breed dogs.

Methods Blood was collected from 172 dogs (152 CKCSand 20 mixed breed) and placed into sodium-citrate anticoagu-lant. Platelet counts were performed manually, by auto analyserand by blood smear estimates in CKCS and mixed breed dogs.Blood smears were also examined for platelet clumping anderythrocyte, leukocyte and platelet morphology. Pedigreeanalysis was performed to determine if an autosomal recessiveinheritance pattern was supported.

Results A statistically significant difference was found inplatelet counts between CKCS and mixed breed dogs(P < 0.0001). CKCS had a platelet count that was 32% that ofthe controls (95% confidence interval, 28 to 37%). There was nosignificant difference between methods used to count platelets.Thirty percent of CKCS had macrothrombocytes. Pedigreeanalysis and examination of obtained and expected segregationratios from 17 CKCS families supported an autosomal recessivepattern of Mendelian inheritance.

Conclusions A high prevalence of idiopathic macrothrombo-cytopenia exists in CKCS in NSW and automated or bloodsmear estimates are sufficient to count platelet numbers. Datasupports an autosomal recessive inheritance pattern.Aust Vet J 2005;83:700-703

CKCS Cavalier King Charles SpanielEDTA Ethylenediamine tetraacetic acid

Platelets are small (1 to 3 µm diameter), anucleate cells thatadhere to damaged blood vessels and participate in primaryhaemostasis.1 They are released by mature megakaryocytes

and circulate in the blood with an average life span in dogs of 5 to7 days.2 In humans approximately 150 billion platelets areproduced each day. Although the normal reference range of 200to 500 x 109/L is broad, each individual maintains a muchnarrower range.3 This requires a fine balance between throm-bopoiesis, platelet senescence and consumption.2 Idiopathicthrombocytopenia with macrothrombocytes has been reported inhumans,3,4 mice5 and CKCS.6-8 It has been shown that CKCS inSweden and Denmark have a high prevalence of idiopathicmacrothrombocytopenia without bleeding tendencies and thatthe syndrome has an autosomal recessive inheritance pattern.6

The degree of thrombocytopenia is usually moderate (50 to 90 x109/L). A similar idiopathic thrombocytopenia without bleedingtendencies has been observed in NSW (personal observations).Practitioners unaware of the syndrome often use immunosuppres-

sive therapy without improvement. The purpose of this study wasto determine the prevalence of asymptomatic idiopathicmacrothrombocytopenia in the population of CKCS in NSW,and to determine if it does exhibit an autosomal recessive inheri-tance pattern. We also aimed to determine if significant differ-ences existed when counting platelets manually, via auto analyseror by blood smear estimation in CKCS and mixed breed dogs.

Materials and methodsBlood was collected in the kennel or show setting from 172 dogs.Of these, 152 were pedigree show dogs of the breed CKCS and 20were of mixed breed. Blood was collected by clean jugularvenipuncture using a 21G, 25 mm needle and 5 mL syringe,placed into a 4.5 mL 3.2% sodium citrate tube and gentlyagitated. Blood smears were made immediately. Manual plateletcounts were performed in duplicate using a haemocytometer(Neubauer; Webster Scientific International, West Sussex,England) and light microscope as described previously.9 Plateletswere analysed by automated counter (Vetscan HMT; Abaxis Inc.Sunnyvale) in 82 CKCS samples and in all mixed breed samples.Platelet clumping, morphology and numbers were estimated byexamination of Diff Quick (Bacto laboratories; Liverpool NSA)stained blood smears from all dogs. Blood smears were also usedto examine erythrocyte and leukocyte morphology. The calcula-tion: one platelet per x100 oil immersion field equals approxi-mately 20,000 platelets/μL was used.1 Presence of macrothrom-bocytes was assessed by counting platelets in ten oil immersionfields. If more than 30% of the platelets were as large as or largerthan erythrocytes the dog was classified as having macrothrombo-cytes.10 All counts were performed within 8 hours of collection.The same person (MKS) performed all counts and was not awareof the source when counting. A normal platelet count was definedas the current standard reference range of 200 to 500 x 109/L.11

Pedigree analysis was performed in order to determine if theinheritance pattern was supportive of an autosomal recessiveinheritance pattern.6 Dogs were classified as affected if theplatelet count was < 200 x 109/L and normal if the plateletcount was ≥ 200 x 109/L.

Statistical analysisStatistical analysis was performed to determine if a significantdifference existed in platelet counts between CKCS and mixedbreed dogs and to determine if there was a significant differencebetween manual, automated and blood smear methods. Thepoint of significance was chosen at <0.05. As data was notnormally distributed it was analysed on the log scale. A mixedeffects analysis of variance was used to investigate the differencesin counts between the two groups of dogs (CKCS and mixedbreed) and to investigate the differences in methods (manual,automated and blood smear). Chi-square test was used to deter-mine if observed numbers of affected CKCS differed from theexpected number. All statistical analyses were performed usingstatistical software (SAS v 8.2; SAS Institute, Cary, NC).

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ResultsAll dogs were healthy, without evidence of bleeding. No dog hadreceived drugs or vaccinations in the 8 weeks before sampling. Nodog exhibited prolonged bleeding from venipuncture sites. Therewere 100 entire female, 2 spayed female, 48 entire male and 2castrate male CKCS. There was 1 entire female, 5 spayed female,no entire male and 14 castrate male mixed breeds. The mean ageof the CKCS was 3 years (range 6 months to 11 years). The meanage of controls was 5 years (range 6 months to 14 years).

The mean manual platelet count was 87.5 x 109/L (95% confi-dence interval [CI] 78.3 to 97.8 x 109/L) for CKCS and 211.1 x109/L (95% CI, 171.3 to 260.2 x 109/L) for controls (Figure 1).Platelet count was not significantly affected by method for eitherCKCS or mixed breed (P = 0.5). One hundred and thirty sevenCKCS (90%) had a manual platelet count of < 200 x 109/L, 83CKCS (55%) had <100 x 109/L and 38 CKCS (25%) had < 50 x109/L (range 16 to 426 x 109/L). No mixed breed dog had aplatelet count of <200 x 109/L (range 214 to 410 x 109/L).

There was a significant difference between CKCS and mixedbreed platelet counts (P < 0.0001, Figure 1 and 2). This was aver-aged across methods because there was no significant differencebetween methods used. Regardless of which method of countingwas used, the platelet count for CKCS was 32% that of the mixedbreeds with a 95% CI of 28 to 37%. The average platelet countfor affected CKCS was 80 x 109/L and for unaffected CKCS was299 x 109/L. For mixed breed dogs the average platelet count was211 x 109/L. There was no significant difference between theaverage platelet count of unaffected CKCS and mixed breed dogs(P = 0.19).

Circulating macrothrombocytes were seen in 46 CKCS (30%)and macrothrombocytes were seen more often in those with aplatelet count of less than 100 x 109/L (70%). No macrothrom-bocytes were seen in mixed breed dogs. No abnormalities inerythrocyte or leukocyte morphology were seen in either CKCSor mixed breed dogs.

Pedigree analysis revealed that there were 17 complete familiespresent in the data set (76 out of the 152 CKCS). A familyconsisted of two parents and at least two offspring. Furtheranalysis of the pedigrees of the 17 families revealed that the inher-itance pattern was most consistent with an autosomal recessiveinheritance pattern as previously reported6 (Figure 3). This wastested by calculating the percentage of affected offspring fromthese 17 families and comparing this to the expected segregationratios for the affected and normal phenotypes. The possiblephenotype crosses present were: affected x affected, normal xaffected and normal x normal. Affected offspring were present inall families when one or both parents were normal, thus all appar-ently normal parents must have been carriers. This resulted inexpected segregation ratios for the three possible phenotypecrosses (affected x affected, normal x affected and normal xnormal) to be 1:0, 1:1 and 1:3 respectively.12 The observed segre-gation ratios were close to the expected ones for the 17 families(Table 1), further supporting an autosomal recessive mode ofinheritance. It was found that 17 of the parents from the 17 fami-lies (61%) were affected, resulting is an estimated gene frequencyof 0.78 (square root of 0.61). Using Hardy-Weinberg law it thenfollows that the expected frequency of carriers was 34% (2 x 0.78x 0.22 x 100) and non carriers was 5% (0.22 x 0.22 x 100).12

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Figure 3. Pedigree supporting an autosomal recessive inheritancepattern in one Cavalier King Charles family.

Figure 1. Mean and 95% confidence limits for platelet countingmethods in Cavalier King Charles Spaniels and mixed breeds.auto = auto analyser, smear = blood smear, manual = manualcount, CL = 95% confidence limit.

Figure 2. Manual platelet counts in Cavalier King Charles Spaniels(n = 152) and mixed breed dogs (n = 20).

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DiscussionPlatelets are essential for haemostasis and maintenance of vascularintegrity. Causes of thrombocytopenia include bone marrowfailure, immune mediated disease, drug toxicity, infectious agents,neoplasia and increased platelet consumption (for example inhaemorrhage and disseminated intravascular coagulation).11

When an unexpectedly low platelet count is obtained, artefact orlaboratory error should also be considered. Platelet aggregationdue to traumatic blood collection, storage and EDTA anticoagu-lant can result in low platelet counts. In humans platelet aggrega-tion and clumping in the presence of EDTA anticoagulantaccounts for 15 to 20% of isolated low platelet counts.3 An accu-rate platelet count can usually be achieved by rapid processing of afresh specimen collected in citrate buffer.3 A similar phenomenonhas been recognised in cats and it has been shown that citrate isthe anticoagulant of choice for platelet counting in this species.13

Hence, sodium citrate was chosen as the anticoagulant in this study.

The prevalence of thrombocytopenia in CKCS in this study was90% with 25% of cases having severe thrombocytopenia (< 50 x109/L). We chose to define the lower limit of normal plateletcount as 200 x 109/L because this was the limit used by commonreferences,1,11 by commercial laboratories in NSW and the lowerlimit set for our impedance auto analyser. A lower limit of 100 x109/L was used by previous studies.6,8 The reason was to morerigorously define those dogs with thrombocytopenia6, 8 however,these authors have also stated that there is, as yet, no clear optimumcut-off value.8 Hence, we did not see a reason to alter the currentnormal lower reference value of 200 x 109/L. If we did use thelower limit of 100 x 109/L the prevalence of thrombocytopenia inCKCS in our study was 55%, which is similar to the prevalence ofthrombocytopenia encountered in CKCS in Denmark andSweden.6,8 Our study was consistent with studies performed inEurope and supported the reports of significant differences inplatelet counts between CKCS and other breeds of dog.

No significant difference existed in counting methods and thiswas consistent for both groups. This was an unexpected finding asit has been previously shown that autoanalysers are not able toaccurately differentiate between large platelets and erythrocytes.10

It is not certain if the underestimation was confined to the auto-mated analyser used in the previous study or all automatedimpedance analysers. Impedance analysers determine plateletcounts within a given size as they pass through an aperture andimpede current across it. Thus, they discriminate betweenplatelets from erythrocytes based on volume.11 When plateletsapproach the size of erythrocytes they will be counted as such.Brown et al10 found that an erroneous diagnosis of thrombocy-topenia was made using automated analysis and suggested thatCKCS platelets be counted manually. This was not found to be

the case in our study. Platelet counts can be affected by traumaticcollection, sample handling, splenic activity, methodology,human error, inherent variation and platelet clumping. In thisstudy, specimen collection, handling and methodology were iden-tical between CKCS and mixed breeds, thus, any error wouldhave been consistent between samples.

No attempt was made to test the effects of age, sex, coat colour orpresence of mitral regurgitation on platelet numbers in CKCS asprevious studies did not find any significant effect of these para-meters on platelet number.6,8,14

Pedigree analysis was in agreement with the findings of Pedersonet al6 in that the inheritance pattern was most consistent with anautosomal recessive pattern of Mendelian inheritance.12 Thesecharacteristics were that parents of affected animals were oftenrelated, generations could be skipped, approximately equalnumbers of male and female dogs were affected and all offspringof two affected parents were affected.12 The high prevalence (90%of dogs affected) implies a low prevalence of non-carriers in thepopulation. No significant difference was found in the averageplatelet counts between unaffected CKCS and mixed breed dogs.It was not possible to draw any conclusions as to whetherheterozygotes differed in platelet count from homozygotes as allunaffected CKCS appeared to be carriers.

The haemopoietic growth factor thrombopoeitin is the primaryregulator of megakaryocyte growth and platelet number. Largenumbers of genes participate in the differentiation ofmegakarycoytes and the subsequent shedding of platelets rangingfrom transcription factors, cytokines, cell surface receptors,signalling molecules, cytoskeleton proteins, molecular motors andcell cycle regulators.3 Mutations in any of these genes could causethrombocytopenia due to a decreased production and shortenedlife span. Although congenital thrombocytopenia in humans wasonce considered rare, these syndromes are now being recognisedwith increasing frequency. The clinical spectrum of the diseaseranges from severe bleeding diathesis in the first few weeks of life(Bernard-Soulier syndrome, Grey Platelet syndrome) to mildconditions that remain undetected through to adulthood(Hegglin anomaly, Sebastian syndrome, Mediterraneanmacrothrombocytopenia).3,4 In the latter group, distinguishingbetween inherited and acquired thrombocytopenia, especiallyimmune mediated thrombocytopenia, is essential to avoid unnec-essary and potentially harmful treatments.3,4

In humans, gene mutation and chromosome location for anumber of inherited thrombocytopenias has been characterised.The syndromes are classified based either on clinical features (ageof presentation, severity, associated abnormalities), platelet size,genetic mutation or inheritance pattern.3 Although most have anautosomal dominant inheritance pattern, some are autosomalrecessive or X-linked. Several have been defined on the basis ofpresence or absence of neutrophil inclusions, macrothrombocytesor associated abnormalities such as nephritis, hearing loss,cataracts and mitral insufficiency.3 The syndrome of thrombocy-topenia in CKCS appears most similar clinically to Mediterraneanmacrothrombocytopenia in humans (lack of bleeding tendency orother abnormalities) however, this syndrome in humans is auto-somal dominant while the disease in CKCS, thus far, appearsautosomal recessive. In humans, the syndrome of Mediterraneanmacrothrombocytopenia has been localised to the short arm ofchromosome 17 with gene mutation of GP1bα.3 Thus far, noattempt has been made to localise the specific genetic mutation inCKCS. The results suggest that CKCS have underlying abnormal-

Table 1. Distribution of affected offspring depending on cross for 17Cavalier King Charles families.

Cross Families Number of Affected Normal % Affected ExpectedOffspringa Offspring Offspring % Affected

Affected x 6 20 20 0 100% 100%affected

Normal x 10 34 20 14 59% 50%affected

Normal x 1 4 1 3 25% 25%normal

aFamilies consist of both parents and = two offspring. All litters had at least oneaffected offspring.

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A high prevalence of thrombocytopenia exists in CKCS in NSW.It is important to consider inherited macrothrombocytopenia inCKCS if clinical signs of bleeding are not present. Veterinariansneed to be aware of the syndrome, that haemorrhagic diathesisdoes not occur and that therapeutic intervention, often withpotentially harmful drugs, is not warranted.

AcknowledgmentsThis work was supported by the Australian College of VeterinaryScientists research grant. The authors would like to thank thenursing staff at the Veterinary Specialist Centre for technical assis-tance and Caro Badcock for the statistical analysis. We would alsolike to thank Rosemary Duffy of the Australian Cavalier KingCharles Association for recruiting participants as well as all dogsand dog owners who participated in the study.

References1. Barger AM. The complete blood count: a powerful diagnostic tool. Vet Clin NAm, Small Anim Pract 2003;33:1207-1222.2. Abrams-Ogg ACG. Triggers for prophylactic use of platelet transfusions andoptimal platelet dosing in thrombocytopenic dogs and cats. Vet Clin N Am, SmallAnim Pract 2003;33:1401-1418.3. Drachman JG. Inherited thrombocytopenia: when a low platelet count does notmean ITP. Blood 2004;103:390-398.4. Mhawech P, Saleem A. Inherited giant platelet disorders. Classification andliterature review. Am J Clin Pathol 2000;113:176-190.5. Novach EK, Reddington M, Zhen L et al. Inherited thrombocytopenia caused byreduced platelet production in mice with gun-metal pigment gene mutation. Blood1995;85:1781-1789.6. Pederson HD, Haggstrom J, Olsen LH et al. Idiopathic asymptomatic thrombo-cytopenia in cavalier King Charles spaniels is an autosomal recessive trait. J VetIntern Med 2002;16:169-173.7. Smedlie LE, Housten DM, Taylor S, Post K, Searcy GP. Idiopathic, asympto-matic thrombocytopenia in cavalier King Charles spaniels: 11 cases (1983-1993).J Am Anim Hosp Assoc 1997;33:411-415.8. Eksell P, Haggstrom J, Kvart C, Karlsson A. Thrombocytopenia in the cavalierKing Charles spaniel. J Small Anim Pract 1994;35:153-155.9. Knoll JS, Rowell SL. Clinical hematology. Vet Clin N Am, Small Anim Pract1996;26:982-983.10. Brown SJ, Simpson KW, Baker S, Spanoletti MA, Elwood CM. Macro-thrombocytosis in cavalier King Charles Spaniels. Vet Rec 1994;135:281-283.11. Feldman BF, Zinkl JG, Jain NC editors. In: Schalm’s veterinary hematology.5th edn. Lippincott Williams and Williams, Maryland, 2000: 443-497, 1057-1063.12. Nicholas FW. Introduction to veterinary genetics. 2nd edn. BlackwellPublishing, Sydney, 2003:25-27,110-114,134-140.13. Tasker S, Cripps J, Mackin AM. Evaluation of methods of platelet counting inthe cat. J Small Anim Pract 2001;42:326-332.14. Cowan SM, Barthges JW, Gompf RE et al. A giant platelet disorder in thecavalier King Charles Spaniel. Exp Hematol (in press).15. Olsen LH, Kristensen AT, Haggstrom J et al. Increased platelet aggregationresponse in cavalier King Charles Spaniels with mitral valve prolapse. J Vet InternMed 2001;15:209-216.

(Accepted for publication 16 August 2005)

ities of the mechanisms that regulate the size and number ofcirculating platelets. While these mechanisms are likely to bethose that influence the maturation and heterogenicity ofmegakaryocytosis, it is possible that they may actually influencelifespan of platelets in circulation or cause preferential sequestra-tion of platelets in peripheral pools.14

In 46 CKCS (30%) platelets appeared large on subjective assess-ment of microscopically examined Diff Quick stained bloodsmears. Canine platelets are generally a quarter to half the size ofan erythrocyte but occasionally can be larger than a red cell.11 Anincreased platelet size can be real or an artefact. Anticoagulant,temperature, storage time, osmotic conditions, and degree of acti-vation may affect the size and shape of platelets. Platelet sizeincreases during accelerated thrombopoeisis (for example inregenerative blood disorders). There are few studies on plateletsize in dogs but large platelets have been found in immune medi-ated thrombocytopenia, bone marrow disease, disseminatedintravascular coagulation, iron deficiency, splenectomy andneoplasia.11 The increased size is thought to be due to increasedstimuli for thrombopoiesis.10 The asymptomatic nature of thethrombocytopenia in CKCS suggests adequate platelet functionwith a greater metabolic activity of macrothrombocytes. This maybe preventing bleeding disorders in the affected CKCS.7 Plateletfunction is thought to depend more on total platelet mass(number x volume) than actual platelet number.7 It is not knownwhy macrothrombocytes occur in some humans and CKCS withcongenital thrombocytopenic disorders, however, it has beensuggested that the macrothrombocytes are probably formed bydefective cytoplasmic blebbing of mature megakaryocytes.3

Platelet function was not specifically tested in the present studyhowever, the absence of petechiae, ecchymosis, bleeding oranaemia suggest normal platelet function. Testing primary coagu-lation function by determining a buccal mucosal bleeding timemay have been a useful way of evaluating platelet function butwas not possible in this study due to blood collection beingperformed in the show and home setting. In vitro platelet aggre-gation studies have been performed in humans3 and CKCS14,15

but conflicting results were obtained in CKCS. One study foundthat CKCS with > 100 x 109/L had increased platelet aggregationin response to adenosine diphosphate and arachidonic acid (bothwith epinephrine) whereas those with platelet counts < 100 x109/L had normal platelet aggregation responses.15 A separatestudy found that all CKCS, irrespective of platelet number, hadsignificantly reduced platelet aggregation.14 It is uncertain as towhy such different results were obtained but differences inmethodology have been suggested as a possible factor.14

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