Dynamic ultrasonography of the hip joint in early diagnosis of canine hip dysplasia

Transcription

1 Dynamic ultrasonography of the hip joint in early diagnosis of canine hip dysplasia Pilot study Veterinary thesis 27 ETCS points Department of Small Animal Clinical Sciences, Faculty of Life Sciences, Frederiksberg Campus, University of Copenhagen, Copenhagen, Denmark. Johan Meilholm Mikkelsen V9209 Thesis Advisor: Dorte Hald Nielsen (DHN) DVM, Ph.D., Assistant Professor Associate Thesis Advisor: Fintan McEvoy (FME) MVB, Ph.D., DVR, DipECVDI, Associate Professor September 2007

2 Preface This veterinary thesis has been prepared in the period from February to September 2007 in cooperation with Dorte Hald Nielsen as the thesis advisor and Fintan McEvoy as co-advisor. The report has been written in relation to the Department of Small Animal Clinical Sciences, Faculty of Life Sciences, Frederiksberg Campus, University of Copenhagen, Copenhagen, Denmark. I would like to express my deepest gratitude to Dorte Hald Nielsen for her excellent guidance and support. She was always available and ready to assist me. Also I would like to thank Fintan McEvoy for his insightful help with programming Image J and the use of the ultrasound equipment. Special thanks to Knud Christensen for patiently guiding me through the world of SAS statistics. Without his help this thesis would have taken much longer to complete. Great thanks to Doctor Charlotte Strandberg for gracefully inviting me to observe the use of ultrasound screening of babies for hip dysplasia in human paediatrics at Gentofte Sygehus. I would like to thank Peter Weis and Steen Engermann and the staff at the Virklund Dyreklinik for their great hospitality in letting me visit their facilities and introducing me to the PennHip method in practical use. Finally special thanks go to my wife Kamilla Meilholm Mikkelsen for her great understanding and care in stressful times. I owe her everything. Copenhagen, September 17 th 2007 Johan Meilholm Mikkelsen

3 Sammendrag Formålet med dette studie var at udforske mulighed for at udvikle en screeningsmetode som kan benyttes til tidlig diagnostik af hofteledsdysplasi hos hund. Metoden gør brug af ultralyd i stedet for ioniserende stråler. Metoden kunne i teorien udføres uden brug af bedøvelse eller sedation. De hunde som indgik i studiet blev evalueret ved 7 og 16 ugers alder med en dynamisk ultrasonografisk metode som er baseret på principperne bag PennHip metoden. Ved 16 ugers alderen blev hundene ligeledes undersøgt med PennHip og Ortolanis metode. Ved 1 års alderen blev hundene røntgenfotograferet med standard ventrodorsal hofteledsoptagelse. Røntgenbillederne blev bedømt af Dansk Kennel Klub. Målingerne son blev opnået ved den dynamiske ultralydsoptagelse blev sammenlignet med resultaterne fra standard røntgenoptagelserne, PennHip og Ortolanis manøvre. HD-indeks for forældredyrene vil blive præsenteret. Resultaterne fra dette studie viser at tidlig diagnostik af hofteledsdysplasi hos hund ved brug af dynamiske ultralydsoptagelser er reproducerbar på både inter- og intraobservatør niveau. Dette studie fandt ikke en signifikant korrelation mellem ultralydsoptagelserne ved 7 ugers alderen og de andre diagnostiske modaliteter. Resultaterne fra ultralydsoptagelserne ved 16 ugers alderen var signifikant korreleret til både PennHip metoden og Ortolanis manøvre. Dog var resultaterne fra ultralydsoptagelserne ved 16 ugers alderen kun korreleret signifikant til den radiografiske standard ventrodorsal hofteledsoptagelse på optagelserne på højre hofte. Yderligere forskning er nødvendig hvis ultralyd skal etableres som en brugbar modalitet til tidlig screening for hofteledsdysplasi hos hund. Det ville være formålstjensteligt at identificere en parameter som er korreleret til størrelsen af hunden. Hvis en sådan parameter kunne identificeres kunne ultralydsmålingerne omregnes til en indeksværdi. Indeksværdien ville så tage højde for størrelsen af hunden og derved ville målingerne blive sammenlignelige mellem hunde af samme race. Størrelsesparameteren behøver ikke nødvendigvis være noget man måler ved hjælp af ultralyd. Diameteren af lårbenet kunne muligvis bruges, men yderligere studier er nødvendige for at vise at diameteren er korreleret til størrelsen af hunden. Kan en sådan parameter identificeres skal der laves referenceværdier for alle hunderacer.

4 Abstract The aim of the present study was to explore the possibility of using a non-invasive test for early screening for Canine Hip Dysplasia using ultrasound that does not require ionising radiation, and which could be performed without medical restraints. The dogs were evaluated at 7 and 16 weeks of age using a dynamic ultrasonographic method based on the principles of the PennHip method. At 16 weeks of age the puppies were evaluated by the PennHip and Ortonlani s method. At one year of age standard ventrodorsal radiographs were taken and graded by the Danish Kennel Club. The ultrasound measurements were compared to standard ventrodorsal radiography, Ortolani s manoeuvre and PennHip. Heritability indexes for parent animals were obtained and are presented. Based on the results of this study the dynamic ultrasonographic evaluation of the canine hip joint in early diagnosis of CHD is a safe, non invasive technique, that does not require ionising radiation. This study showed no significant correlation between the results of the ultrasonographic evaluation at 7 weeks of age and the other diagnostic modalities. However the results from the ultrasonographic evaluation at 16 weeks of age were correlated with both PennHip and Ortolani s manoeuvre. The ultrasound measurements at 16 weeks were only correlated to standard ventrodorsal radiography on the right hip. The procedure is reproducible. The inter- and intraobserver variance analysis showed no statistically significant variation in measurements done by the two observers. Further research is needed to establish ultrasound as a credible modality for hip dysplasia screening programs. Firstly a reference measure for the size of the dog should be identified. By identifying such a measure the distraction distance measurements could be converted into an index. The index would then take the size of the dog into account so as to make the measurements comparable between dogs of the same breed. As suggested the reference marker does not have to be something measured by ultrasonography. To establish if the diameter of the femoral shaft could be used as reference marker, studies would have to be undertaken. If a reference marker is identified, then the next step would be to determine reference values for normal and dysplastic hips. Without a reference marker, ultrasound measurements of every breed at every age, both normal and dysplastic, would have to be established. This is not a viable solution.

5 Contents 1. Introduction Literature study Aetiology Pathogenesis Screening - Methods Standard ventrodorsal radiography Ortolani s manoeuvre Hip dysplasia selection index PennHip Ultrasound imaging Materials and methods Hip dysplasia selection index Ultrasound imaging Analysis of ultrasound images Ortolani s manoeuvre PennHip Standard ventrodorsal radiography Results Data Hip dysplasia selection index Ultrasound measurements Ortolani s manoeuvre PennHip Standard ventrodorsal radiography Statistics Interobserver variance Intraobserver variance Correlation coefficients: Standard ventrodorsal radiography Discussion Conclusion Perspective Ultrasound in the future Gene test References Page 1 of 36

6 1. Introduction Canine hip dysplasia (CHD) was first described in 1937 by Gerry B. Schnelle. In a paper entitled Congenital Subluxation of the Coxofemoral Joints of a Dog Schnelle writes: "The condition described herein, rare though it may be, should be recognized as being congenital and potentially hereditary, and the dog or bitch in which it occurs should be destroyed or sterilized in the eugenic interests of the breed." (1). Schnelle s description was modified in 1966 by Henricson, Norberg and Olsson as: "A varying degree of laxity of the hip joint permitting subluxation during early life, giving rise to varying degrees of shallow acetabulum and flattening of the femoral head, finally inevitably leading to osteoarthritis." (2) Canine hip dysplasia is the term used to describe any disease complex that results in osteoarthritic change in the coxofemoral joint. It is important to remember that hip dysplasia as such can be caused by any disease that gives rise to laxity during early life, e.g. myelomeningocele, brain damage and poliomyelitis. However hip dysplasia of this type is of little significance in the dog (2). Degenerative radiological changes of the hip joint similar to those of CHD can present as an acquired disease. Later development of osteoarthritis and degenerative bone disease can be due to obesity (3), excess exercise (3), trauma i.e. fractures and luxation or malformation of pelvis and columna i.e. transitional vertebrae. In the following the term is reserved for the developmental abnormality that results from early life joint laxity. At present all screening methods for CHD is based on radiography (4;5). The procedures all benefit from deep sedation or general anaesthesia to produce optimal results (6). The purpose of this study is to explore the possibility of using a non-invasive test using ultrasound that does not require ionising radiation, and which could be performed without medical restraints. Ultrasonographic measurements will be compared to standard ventrodorsal radiography, Ortolani s manoeuvre and PennHip. Heritability indexes for parent animals will be presented. Page 2 of 36

7 2. Literature study 2.1 Aetiology From the very beginning a hereditary component of canine hip dysplasia was assumed (1). However attempts to explain the inheritance of hip dysplasia using ordinary Mendelian modes did not sufficiently fit the data observed. Nevertheless, both recessive and dominant modes of inheritance were proposed. Later on, it was proposed that CHD was determined in a multifactorial way (7). Recently, this thesis was supplemented and refined, as a major gene was detected as a cause of CHD in addition to a polygenic component (8). At present, projects are under way with the aim to locate quantitative trait loci (QTL) significantly linked to CHD, and ultimately to develop genetic tests to identify carriers of genes responsible for CHD (8-10). But genetics alone can not explain the triggering of the disease. Not all dogs that have the predisposing genotype express CHD in their phenotype. Several environmental factors have been identified as triggers for CHD. (2;11) Feeding seems to play an important role (12), as dogs fed a diet that promotes rapid growth have a higher prevalence of CHD than dogs fed a restricted diet (13-16). This is not only true post-weaning, but also intake of breast milk in excess increases the prevalence of CHD (17). The mineral balance of the feed also has an influence, especially calcium content. Both excess and deficiency of calcium had deleterious effects. (18;19) Because of the environmental influence on the presentation of CHD, the heritability of the disease is low, ranging from 0.2 to 0.6 (11;20;21). Some breeds are more susceptible than others (22-27). There is a difference in prevalence according to size, as larger breeds are more prone to CHD (23;25). Late ossification of the femoral head has been observed in breeds that are more prone to CHD (28), though later research did not find the same difference (29). Page 3 of 36

8 This difference in genotype versus phenotype makes CHD very difficult to eradicate. Because of the environmental influence, genetically predisposed individuals may not present with CHD in routine screening programs, constituting false negatives. Therefore they will be regarded as free of the disease in screening programs and propagate CHD-genes onto their offspring. (2;11;30) 2.2 Pathogenesis By definition, dysplasia is an abnormality of development; in pathology an alteration in size, shape and organisation of adult cells. In this context the interpretation of dysplasia is one of faulty development as changes occur during the postnatal remodelling of the bones. It is not a prenatal fault in development of the acetabulum as is the case in humans (31). Canine hip dysplasia is a developmental abnormality. Puppies are born with normal hips (4;5). Instability of the coxofemoral joints develops during the postnatal remodelling of the bones. It is generally agreed that the initiating factor is joint laxity (2;4;5;26;32;32-37). Joint laxity starts a vicious circle: Movement and weight bearing forces the femoral head out of its normal position within the acetabulum. The growth and formation of the acetabulum and to some extent of the femoral head is seriously affected. Incongruity of the joint leads to the secondary changes associated with CHD. The acetabulum becomes flattened and the femoral head looses its ball-like structure as a result of the forces applied. The cartilage of the hip joint often proliferates and may be associated with periosteal proliferation (2). 2.3 Screening - Methods Both national and international organisations are involved in the screening programs that have been set up to bring the prevalence of CHD in the population down. Although different classification systems are used, there is a general consensus in the choice of procedure. The most widely used is the ventrodorsal radiographic projection, though other alternatives do exist. (38-40) Page 4 of 36

9 2.3.1 Standard ventrodorsal radiography The standard ventrodorsal radiography is the Gold Standard for diagnosis of CHD worldwide. Both the Fédération Cynologique Internationale (FCI) (appendix A) and the Orthopedic Foundation for Animals (OFA) (41) recommends this procedure for screening programs. In the following only the FCI protocol is used since the Danish Kennel Club (DKC) is a member of FCI and it is the DKC who have been involved in the grading of the radiographs obtained in this study. The FCI has set forth guidelines for evaluation of hip joints in regards to classification of hip dysplasia. The guidelines state that a dog can not be submitted for approval before years of age depending on the breed. It is possible to detect joint laxity by this method, but it may be masked in the ventrodorsal radiograph due to rotational tightening of the round ligament in the hip-extended position (36;42). The age limit of 12 months or older is set, so that the diagnosis can be made on the basis of secondary changes, which should be present at this age (43). The sensitivity of the test is very poor at a young age. In a population of German Shepherd dogs it was shown, that the sensitivity of standard ventrodorsal radiography in relation to age of examination, that is to say the radiographs gave a correct diagnosis of CHD, was as follows: 15% at 6 months, 69% at 12 months and 95% at 24 months of age (44). Standard ventrodorsal radiography is performed as explained in this paragraph. The dog is placed in dorsal recumbency preferably in a foam wedge or other types of support. The pelvis should be parallel to the table, and should be symmetric. The hind limbs are drawn caudally and the femurs should be parallel to one another and to the table. The hind limbs are pronated so the patellae overlie the femoral trochleas. The inward rotation of the stifles insures that the femoral neck is clearly visible. The x-ray beam is centred at the level of the coxofemoral joints. (4) Page 5 of 36

10 For an example at a correctly positioned radiograph see figure 2.1. It has been shown that anaesthesia has an effect on the result of evaluation of the radiographs taken by this method. There is a significant increase in the prevalence of CHD, when the dogs are anaesthetized compared to when they are not. The Norberg angle was significantly decreased when the dogs were anaesthetized (6). The radiograph is graded on a scale from A to E. For detailed description of the individual grades see appendix A. Standard ventrodorsal radiography relies on a subjective evaluation of the radiographs. This gives rise to problems with a low degree of interobserver agreement with the method. This was underlined in a recently published article. It showed interobserver Figure 2.1 Example of correct positioning for the standard ventrodorsal radiography agreement, when observers were asked to classify a (Courtesy of Dorte Hald Nielsen) dog as dysplastic or non-dysplastic, was 72%. When observers were asked to grade a dog using the A-E system the interobserver agreement was 44%. The study did however conclude that although the interobserver agreement was low, the agreement did increase with experience (45) Ortolani s manoeuvre Ortolani s manoeuvre was originally developed for human diagnostics of hip dysplasia. This test is based on manual detection of joint laxity (46). It has been adapted for use in dogs (47). The dog is placed in dorsal recumbency. A helper steadies the front part of the dog. The examiner grasps the stifles and holds both femurs perpendicular to the table. Whilst holding one leg in this fixed position, the other leg is abducted and a slight downward pressure is applied (fig. 2.2). Page 6 of 36

11 Ortolani s manoeuvre can be performed in the conscious dog but it is best performed under anaesthesia in the clinically affected dog. If joint laxity is present, the head of the femur will ride up onto the dorsal rim of the acetabulum, and then, as the Figure 2.2 Ortolani s maneouvre. By applying downward pressure the coxofemeral joint can, if lax, be subluxated. When the leg is abducted limb is abducted, it will fall the femoral head will slide over the acetabular rim producing the back into the acetabular clunk. (47) fossa with a palpable and sometimes audible "clunk". This is called the Ortolani s sign and can be elicited in either dorsal or lateral recumbency by holding the femur in the neutral position for adduction/abduction and compressing the hip joint by applying pressure to the flexed stifle joint. When performed in lateral recumbency, the other hand is placed over the dorsal aspect of the pelvis to provide a counter force. The table provides this force when the test is performed in dorsal recumbency. The nature of the "clunk" associated with subluxation and reduction of the femoral head can give additional information about the hip joint conformation. A "soft clunk" on reduction suggests that the dorsal acetabular rim may have been damaged by repeated subluxation during weight bearing, involving microfractures and flattening of the dorsal rim. Damage to the dorsal acetabular rim is highly significant as weight bearing is concentrated in this area of the acetabulum. (48) Although hip joint laxity is an integral part of hip dysplasia, not all dysplastic dogs will have palpable hip laxity. This is particularly true in the older dog as secondary changes including joint capsule thickening and periosteal proliferation will act to reduce joint instability. Therefore a negative Ortolani s manoeuvre does not exclude the diagnosis of hip dysplasia. (48) Page 7 of 36

12 2.3.3 Hip dysplasia selection index Several studies have shown, that the frequency of CHD can be reduced by individual selection i.e. selection based on the phenotype, that is the degree of hip dysplasia by standard ventrodorsal radiography, of individuals (20;25). However, even within less well-controlled screening programmes, the frequency of CHD can be reduced more effectively by using a better breeding value. For this purpose the hip dysplasia selection index (HD-index) was developed as an adaptation from breeding programmes for production animals i.e. breeding values. The HD-index was introduced in Denmark in 1985 and has since been used by the DKC. For every breed of dog the mean index value is set at 100. This means that the mean index value is not static. The index value 100 changes every time a new dog has its CHD results registered. (49) HD-Index values are calculated for each individual dog on the basis of the dog and its parents, siblings and offspring. The testing of offspring is the most influential variable. The HD-index value therefore is a statistical measure of the individual dog s gene pool in regards to the breed prevalence of CHD. As more family members especially offspring are taken into account when calculating the HD-index the power of the individual indices increase PennHip Pennhip stands for Pennsylvania Hip Improvement Program. This method was developed at Pennsylvania State University by Gail Smith. He felt that it was necessary to find an alternative to the standard ventrodorsal hip-extended radiograph, due to the amount of falsenegative diagnoses (5-8%) that this method produced (40;44). This was partly due to the tightening of the round ligament during the hip-extension and also the subjectivity of the method. Also the fact that a definitive diagnosis could not be made before 2 years of age (OFA standards) was of great concern. It would be of great benefit if the diagnosis and subsequent breed approval could be established at an earlier age, preferably before sexual maturity. This would save breeders time and money in the selection process towards reducing the incidence of CHD. (36) Page 8 of 36

13 The basis of the PennHip method is that joint laxity is the first sign of hip dysplasia, and that it precedes the secondary bone changes. The passive laxity is measured by a dynamic stress radiography of the hip joints (27;35;37;40;50). The procedure is considered valid from 16 weeks of age (51). PennHip is a commercial test method that requires American authorisation, and all radiographs have to be graded in the USA. (52) With this method, hip laxity of each dog is evaluated radiographically in three positions. 1) The standard ventrodorsal, hip-extended position of the pelvis. 2) A compression position radiograph, which simulates weight bearing as if the dog was standing. The dog is positioned in dorsal recumbency, the stifles are grasped, and the femurs are positioned perpendicular to the tabletop. To assure that the femoral heads are fully seated within the acetabulae pressure is applied laterally. 3) A distraction view is made with the dog in dorsal recumbency, using an adjustable distraction device positioned between the dogs distended legs (fig. 2.3), firmly pressed down upon the pelvis. While the tarsi are grasped the stifles are pushed together using the distraction device as a fulcrum to impose a lateral distraction force between the femoral Figure 2.3 Use of distraction device in PennHip heads and acetabulae. ( The degree of excess laxity between the femoral head and acetabulum allows diagnosis of impending hip dysplasia in advance of bone changes. (5) The distance that the femoral heads can be displaced by distraction, compared to compression, is the distraction distance. This is measured by overlaying the compression and distraction radiograph. Page 9 of 36

14 It has been shown, that anaesthesia has an effect on the evaluation of the radiographs taken by this method. There is a significant increase in the amount of joint laxity present when the dogs are anaesthetized compared to when they are not. (6) The quantitative measurement of hip joint laxity the distraction index (DI), is correlated with the size of the femoral head via the radius (fig. 2.4). The breed of the dog has to be taken into consideration when interpreting the result, as it has been shown that there is significant interbreed variation in the probability of developing CHD and the DI (27). Figure 2.4 Distraction index (DI). Red circle: Femoral head at compression. Yellow circle: Femoral head at As a rule of thumb a DI < 0.3 indicates distraction. d: Distance between the centres of the femoral heads at compression and distraction. r: radius a low probability of developing CHD, of femoral head, measured at distraction. ( whereas a DI > 0.7 indicates a significant probability of developing CHD. A DI between 0.3 and 0.7 is grey zone. Validation of this method has shown that the reliability in clinical settings is good (53) Ultrasound imaging Ever since hip dysplasia was recognised in the dog, there has been an interest in seeing if the condition was comparable to the human counterpart so that the dog could be used as a spontaneous animal model (25). The dog as a model of human hip dysplasia There are, however, several differences that distinguish canine from human hip dysplasia. First and foremost canine hip dysplasia is a developmental abnormality, whereas human hip dysplasia is a congenital condition. The aetiology of human hip dysplasia is believed to be partly genetic, but other factors are involved such as intrauterine positioning. Babies, that are born in breech position have a higher prevalence of hip dysplasia, than those born in the Page 10 of 36

15 normal position (46). In dogs intrauterine position has no significance in regards to the prevalence of CHD. The foetal hind limbs are short and not prone to dislocation, and the puppies have normal hips at birth (5). Also there is a significantly higher prevalence in girls than boys (54;55), whereas in the dog there is no significant sex predisposition (22-24;26;27). The hip joint of a puppy ossifies from 8 weeks of age (4), which is much faster than human babies. This becomes a challenge in respect to ultrasound evaluation of the hip joints in dogs. The use of ultrasound in human hip dysplasia The ultrasonographic modalities have long been established in human medicine as the preferred diagnostic tool for hip dysplasia. Screening of newborns is made on the basis of positive palpatory signs of joint laxity at birth. Ultrasound evaluation is undertaken to confirm the presence of laxity and preventative measures are taken if laxity is present. No sedation is required and the procedure is completed in a matter of minutes. (46;56;57) The procedure consists of two parts. A measurement of intraarticular angles is performed. Objective assessment can be achieved by measuring Graf's alpha and beta angles. The alpha angle is the angle between the straight lateral margin of the ilium and a line drawn from the inferior point of the iliac bone tangential to the bony acetabulum (fig. 2.5). The alpha angle reflects the depth of the bony acetabular roof and in a mature hip will be more than 60. A small alpha angle indicates a shallow bony acetabulum. The beta angle is the angle between the straight lateral margin of the ilium and a line drawn from the bony rim to the middle of the acetabular labrum (fig. 2.5). The beta angle assesses the cartilaginous acetabular roof and in a normal infant will be less than 50. An increased beta angle reflects superior displacement of the femoral head. The beta angle is useful predominantly in the subclassification of dysplastic hips. (58) Figure 2.5 Graf s hip angle measurement. ( The stability of the hip can be assessed by internal rotation and adduction of the hip (Barlow s manoeuvre), whilst Page 11 of 36

16 scanning in the standard imaging plane. If the femoral head is dislocated in an anterior or posterior direction, the femoral head will not be visualized in the standard imaging plane of the acetabulum and the transducer will have to be moved to locate the dislocated femoral head. (58) The use of ultrasound in evaluating the stability of human hip joints in children has been shown to be accurate up to the age of two years. (46;59). Ultrasound in canine hip dysplasia Ultrasound is valuable for assessing joint disease. Joint effusion, thickening of the joint capsule and cartilage defects can be identified sonographically. It is also possible to detect bone destruction. Instabilities are often identified by dynamic ultrasonographic examination. (60) Ultrasound of the hip joint of the canine neonate was first described in In this study sonographic imaging of the hip joint was performed on deceased 7-8 weeks old puppies. It concluded that refinement of techniques was necessary, if ultrasound was to be useful in veterinary diagnostics. (61) In 1992 a new study showed, that the anatomy of the hip joint could be identified at up to eight weeks of age. This time both live and dead puppies were used. Most importantly motion of the femoral head within the acetabulum could be observed. Also measurements of the acetabulum, femoral head and the joint space could be made, though they were inconsistent. Several different views were evaluated. At twelve weeks ossification prevented complete hip joint evaluation. In this study no anaesthetics or sedatives were necessary for the ultrasound examination. (62) In the following search for an ultrasonographic method of evaluating the canine hip joint for early signs of CHD two main approaches has been used. One method is to measure angles as in human medicine, the other is an attempt to apply the principles of the PennHip method by measuring joint laxity as distraction distances, but by using ultrasound instead of x-rays. Page 12 of 36

17 Measuring angles The preliminary findings of the first two studies, done with ultrasound on the canine hip joint, prompted further investigation into the use of this modality for detecting early signs of canine hip dysplasia. One study adopted the human approach and measured alpha and beta angles in 2-4 week old Labrador puppies. Their findings were inconclusive (63). A similar study on 2 week old German Shepherds showed that ultrasound was capable of displaying the anatomy of the hip, but due to poor follow-up, no significant results were obtained (64). A recently published article by Flöck et al. re-examines the human approach by measuring alpha and beta angles. Sonographic examinations were carried out on 78 hip joints in 38 dead and one living germane shepherd puppy (1 day - 8 weeks old). In order to show sonoanatomy and sonomorphological development, 24 hip joints were prepared and examined ultrasonically in water. The authors found, that it was generally possible to identify anatomical features sonographically from the first day of life onwards. As in previous studies the ossification centre of the femoral head was a limiting factor in this respect, since its structures could only be seen distally up to the 8th week. A standardised sonographic examination with the corresponding findings is reported. The ossified shape of the hip socket, the cartilaginous overlap of the acetabular cavity and the looseness of the joint are determined objectively. For this purpose both angle alpha and angle beta, as well as the distraction value are fixed as an index. The first reference values and maturity curves have been determined. (65) Measuring laxity Unpublished work by Barbara Gosink et al. describes several different modalities for evaluation of the canine hip joint. 60 puppies were examined at 8 weeks and at weeks of age. They found, that the most useful view was the transverse adduction with manipulation of the hip joint. The preliminary results were that hip joint laxity could be identified and measured, and that these findings correlated with their PennHip results. Again no sedation was required to obtain useful ultrasound images. (66) A similar approach to dynamic ultrasonographic evaluation of passive coxofemoral joint laxity was used to examine a total of 30 puppies. Both greyhound (n = 13) and non-greyhound (n = 17) puppies were used. Puppies were evaluated sonographically at 4, 6, 8, 12, 16, and 26 weeks of age. The coxofemoral joints were distracted manually, and the femoral head Page 13 of 36

18 displacements were measured during distraction. Puppies between 6 and 16 weeks of age yielded high-quality images of the components of the coxofemoral joints. Analysis of the images, including blending of images and all measurements, was performed with image manipulation software. Attempts were made to measure the size of caput femoris, but it was not possible to obtain consist measurements. The greyhounds had significantly smaller maximum distraction distance than non-greyhound puppies. In six- to eight-week-old puppies, the maximum distraction distance was correlated significantly (p of , r 2 of 0.27) with stress radiographic indices. Coxofemoral joints interpreted as being abnormal on hip-extended radiographs taken at one year of age were associated significantly (p of ) with higher maximum distraction distances in six- to eight-week-old puppies. (67) A follow-up on this study was done in This time the use of distraction devices was implemented. A small adjustable distraction devise was placed between the proximal thighs to assist manual distraction of the hips. Hip joint laxity was evaluated in Golden Retriever (n = 60), Labrador Retriever (n = 23), and Labrador/Golden Retriever mix (n = 24) puppies. Three dynamic ultrasonographic measurements were used. Each puppy was evaluated twice; at 6.5 to 9 and 43 to 79 weeks of age. These methods were compared for accuracy in predicting the development of canine hip dysplasia with or without degenerative joint disease by a median age of 16 months. Ultrasound measurement was a reliable predictor of canine hip dysplasia/degenerative joint disease for Labrador-Golden Retriever mix puppies, but was not reliable for the other two breeds. Ultrasonographic methods of evaluating hip joint laxity in puppies at 6.5 to 9 weeks of age were not consistently reliable for all three breeds in predicting hip dysplasia with or without degenerative joint disease at one year of age respectively. Comparing the maximum distraction distance at 7.3 weeks of age with incidence of degenerative joint disease at 65 weeks of age, distraction distances of less than 2 mm, 2-2.9, 3-3.9, and 4 mm or greater resulted in degenerative joint disease incidence of 9/60 (15%), 3/36 (8.3%), 7/40 (17.5%) and 10/76 (13.2%), respectively. Mean distraction distance from the three measurements taken of each hip without using the distraction device, one operator (using the device) and two operators (using the device) distraction distances were also analyzed for relationship with the incidence of degenerative joint disease at 65 weeks. Maximum distraction distance, mean distraction distance and one operator (using the device) measured using dynamic ultrasonography correlated to strict assessment of canine hip dysplasia for Labrador-Golden Retriever mix hips only (p = ). None of the dynamic ultrasonography Page 14 of 36

19 measurement results were significantly related to degenerative joint disease or strict assessment of canine hip dysplasia for all dogs. (68) In 2001 Ohlerth et al. used the PennHip method combined with ultrasound. The dogs evaluated in this study had a mean age of 244 days. Dynamic ultrasonography of the coxofemoral joints was performed during general anaesthesia. The dogs were positioned in a U-shaped trough in dorsal recumbency. Dynamic ultrasonography was performed by two operators, one controlling the transducer, and the other distracting the coxofemoral joints using the PennHip distractor and the PennHip method. For this, the transducer was positioned in between the distractor bars on one coxofemoral joint, directed in a mildly oblique caudomedial to craniolateral plane. The oblique positioning of the probe was considered important for visualisation of the craniolateral acetabular rim as a landmark for the measurements. Images were taken in the non-stressed position and at the end of maximum distraction. On the longitudinal images, the geometric centre of the femoral head was determined with a circular stencil. On both oblique images, with the joint either in the nonstressed or distracted position, a tangent was drawn from the craniolateral acetabular rim to the femoral head. The geometric centre of the femoral head and the tangent of the joint in the non-stressed position were transferred to the distraction image. Then, a vertical line was drawn from the centre of the femoral head to each tangent. The distance between the intersept points on the tangents (the so-called distraction distance) was measured. The ultrasonographic distraction index (DI) was derived by dividing the distraction distance by the radius. Longitudinal and oblique images with the joint in the non-stressed or distracted position were obtained for three repetitions per joint. The maximum value of the three measurements was used for subsequent analysis. Correlation between PennHip and ultrasonographic DI was significant (P< and P<0 0001) but low (r=0 28). At the level of each joint, correlation coefficients were very similar. Sensitivity and specificity were moderate for ultrasonographic DI. (52) In 2007 the results from a Brazilian study based on static ultrasonography, conventional radiography (CR) and distraction radiography (DR) methods were published. The study involved 22 hip joints of Whippet (n=5), Rottweiler (n=4) and Labrador Retriever (n=2). The young dogs were evaluated with the aim of comparing the ultrasonic examination of coxofemoral joints with the radiographic evaluations, both distraction and conventional procedures, for the early diagnosis of passive laxity. Page 15 of 36

20 The ultrasonographic image taken at the age of 14 days allowed the observation of detailed anatomic features, with easy manipulation of puppies. In this study it was not possible to visualize the entire triradiate cartilage after 15-day-old, due to the acoustic shadow produced by the ossification center of the femur head. The study showed, that the static ultrasonographic evaluation was not sensitive in demonstrating a passive articular laxity in 14-day-old dogs. Even with the apparent normal anatomic conformation, there is a possibility of occurrence of laxity, which is not visualized through an ultrasonographic examination, unless the femur head is forced to come out of the acetabulum. The conclusion was therefore, that there is a need for a dynamic ultrasonographic evaluation. (69) Page 16 of 36

21 3. Materials and methods The practical part of this study was done in 1997 and 1998 by Dorte Hald Nielsen (DHN) and Jens Sejer Madsen (JSM). For this study 25 German Shepherds from 7 litters were included. Both sexes were represented equally with 12 females and 13 males. The litter size ranged from 2 to 4 puppies. (5 litters n=4, 1 litter n=3 and 1 litter n=2). The breeders who supplied the litters were chosen at random from a list supplied by the Danish German Shepherd Club. The puppies were examined at 7, 16 and 52 weeks of age. 3.1 Hip dysplasia selection index The hip dysplasia section index for all parent animals are available to the public and were obtained from the DKC. A few of the parent animals had heredity indexes from Germany. These were converted to Danish hip dysplasia selection indexes. 3.2 Ultrasound imaging The approach chosen for this study is a dynamic evaluation of the hip joint laxity. This modus operandi is inspired by the PennHip method. Puppies were sedated with a solution of Combelen (propionylpromazine, Bayer), Atropin (Atropinsulphate, KVL-Apotek) and Metadon DAK (methadonchlorid, Nycomed Danmark) given as a intramuscular injection. After 10 minutes the puppies were given Domitor Vet (Medetomidin, Orion Pharma) intramuscularly and placed in lateral recumbency, with legs in a neutral position. Isopropyl alcohol and coupling gel were applied in copious amounts (clipping of the hair was not possible due to lack of compliance from both breeder and owner). An ultrasound machine, B&K 3535, with an 8 MHz linear transducer was used. The transducer was placed cranially to the great trochanter, perpendicular to the columna (lateral transversal section) (fig. 3.1) and the deepest point was located. The transducer was the rotated around this point until the ileum was horizontal. Page 17 of 36

22 With the transducer fixed in this position the assistant wrapped his fingers around the medial aspect of the femoral shafts and thumbs on the greater trochanter. The coxofemoral joint then was manipulated so that the femoral head was first forced into the acetabulum, thereby fully compressing the joint (fig. 3.2). Maintaining the transducer in the fixed position the assistant displaced the femora laterally to cause lateral subluxation, by distraction to the limit of the passive joint laxity (fig. 3.3). Figure 3.1 Drawings of the ultrasound transducer placement on the hip as viewed from rear (left) and side (right). (67) Fig 3.2 Fig 3.3 Figures 3.2 and 3.3 Ultrasonographic images of the coxofemoral joint of a 17 week old German Sherperd in compression (3.2) and distraction view (3.3). The desired images display the craniodorsal ilium as a horizontal, near-straight line and the rounded femoral head should be clearly visible. The puppies were examined at 7-8 weeks and weeks of age. Each observer performed three scans of each hip at both age intervals. The puppies were examined in random order. It was also randomised which of the two observers (DHN or JSM) was the first to examine each individual puppy Analysis of ultrasound images Ultrasound images obtained were digitized into JPEG-format. A video camera recorded the display monitor of the ultrasound machine, whilst the dogs were scanned. During the scan the Page 18 of 36

23 capture frame function was used when the desired views were obtained. Playing back the recorded video on a television set it was possible to photograph the television screen with a digital camera. The pictures were finally transferred to a computer. Analyses of images were performed with image manipulation software Image J freely available from (70). The images were compared pair-wise. A macro was written that allowed output readings of specific coordinates designated by the user (see appendix B). Coordinates for pelvis caput femoris - distraction caput femoris - compression fix point on the iliac crest (x 0,y 0 ) and femoral head in either compression (x 1,y 1 ) or distraction (x 1,y 2 ) were used to calculate an imaginary point x 0, y 0 x 1,y 0 h 1 h2 x 1,y 2 x 1,y 1 Difference in height is calculated (x 1,y 0 ). These three points together form a right angled triangle (fig. 3.4). Figure 3.4 The the distance that the femoral head could be displaced is measured as the difference in heigts h 1 -h 2. Fix point, pelvis Fix point, cf - dis Fix point, cf - comp Imaginary punkt Calculating the height of the triangles in the distraction view and compression view, as well as the difference in the height of the two triangles was done using an Excel work sheet (Microsoft Corp., USA). The difference in height, measured in centimetres, was the final output data from this procedure. Pixel per cm was calculated as the distance in pixels between two cm-markers on the basis of their coordinates (fig. 3.2 and 3.3). This was done for every image individually. The maximum distraction distance of the three measurements for each hip at each age, were used in subsequent analysis. 3.3 Ortolani s manoeuvre Ortolanis manoeuvre was performed at weeks of age by one observer (JSM). The procedure was carried out while the puppies were sedated. Page 19 of 36

24 Each hip was assigned a score based on a subjective scale from 0 to 3, 0 being tight hips, 3 indicating significant palpable subluxation. 3.4 PennHip Both observers are certified to perform PennHip and both took part in this procedure which was carried out at 16 to 18 weeks of age on all puppies. The radiographs were sent to the Symbiotic Cooperation, owners of the PennHip-method, for grading. 3.5 Standard ventrodorsal radiography At 12 months of age standard ventrodorsal radiographs were taken of the hip joints of the puppies. The radiographs were submitted blinded to the Danish Kennel Club (DKC) for grading. Owner compliance was low which resulted in only 15 out of 25 dogs being submitted for FCIgrading at 1 year. Page 20 of 36

25 4. Results 4.1 Data All data is available in appendix C Hip dysplasia selection index Hip dysplasia selection indexes for all parent animals were obtained. The minimum value was and the maximum value was Ultrasound measurements 22 dogs were examined at 7 weeks of age and 3 dogs were examined at 8 weeks of age. These have been pooled together in a group called 7 weeks. 13 dogs were examined at 16 weeks of age, 4 dogs 17 weeks of age and 8 at 18 weeks of age. These were pooled together in a group called 16 weeks. At 7 and 16 weeks 3 measurements were made by each examiner of each hip. A total of 1200 ultrasound images should have been produced. One observer (DHN) is missing observations on 3 dogs at 7 weeks; the other observer (JSM) is missing observations on one dog at 7 weeks. This brings the images total to The minimal distraction distance measured was 0.01 cm and the maximal distraction was 1.00 cm Ortolani s manoeuvre 4 dogs did not have Ortolani s manoeuvre performed. The scoring ranged from 0 as minimum to 3 as maximum. Page 21 of 36

26 4.1.4 PennHip PennHip was performed on all dogs. The minimum DI was 0.20 and the maximum DI was Standard ventrodorsal radiography 10 dogs did not receive a FCI-evaluation at 1 year of age. The remaining 15 dogs had grades ranging from minimally A and maximally E. 4.2 Statistics Statistical calculations were made using SAS Interobserver variance The GLM (general linear model) procedure was used to determine the variance between the two Table 4.1 Interobserver variance. F-values. None of the F-values are significant (p > 0.05). Observer observers JSM and DHN. Calculations were done on ultrasound measurements of both right and left hip, using only the maximal distraction distances of Left hip Right hip each measurement (App. D). Results are displayed in table 4.1. The H 0 : The mean maximal distraction distance measured by JSM and DHN is the same. H A : The mean maximal distraction distance measured by JSM and DHN is not the same. Since the F-values calculated are not significant at a 95% confidence level H 0 can not be rejected. From this follows that there is insufficient evidence to conclude that there is any difference between the ultrasound measurements made by JSM and DHN. The observed differences in the mean distraction distances in this study can readily be attributed to chance variation. Page 22 of 36

27 4.2.2 Intraobserver variance Again GLM was used to determine the variance within the measurements of the observers JSM and DHN. Calculations were done on all the ultrasound measurements of both right and left hip (App. E). Results are displayed in table 4.2. Table 4.2 Intraobserver variance. F-values. None of the F-values are significant (p > 0.05). Observer Left hip 0.25 Right hip 1.48 The H 0 : The mean distraction distance measured at the three measurements done at 7 and 16 weeks of age by JSM and DHN is the same. H A : The mean distraction distance measured at the three measurements done at 7 and 16 weeks of age by JSM and DHN is not the same. Since the F-values calculated are not significant at a 95% confidence level H 0 can not be rejected. From this follows that there is insufficient evidence to conclude that there is any difference within the ultrasound measurements made by JSM and DHN. The observed differences in the mean distraction distances in this study can readily be attributed to chance variation Correlation coefficients: Pearson Correlation Coefficients were calculated for the entire data set in respect to age using the CORR procedure (see App. F). The correlation between ultrasound measurements and PennHip, and ultrasound measurements and Ortolani s manoeuvre for 7 weeks and 16 weeks of age are displayed in table 4.3 and table 4.4 respectively. At 7 weeks only one correlation coefficient was significant at p < At 16 weeks all correlation coefficients were significant at p < Table 4.3 r-values at 7 weeks of age. *significant at p < weeks Table 4.4 r-values at 16 weeks of age. ** significant at p < weeks PennHip vs. ultrasound PennHip vs. ultrasound Ortolani s vs. ultrasound Left hip Right hip * Ortolani s vs. ultrasound Left hip 0.36** 0.52** Right hip 0.59** 0.45** Page 23 of 36

28 4.2.4 Standard ventrodorsal radiography Statistics on the categorical data obtained by the grading of the standard ventrodorsal radiography were made using the GLM procedure combined with Duncan's Multiple Range Test (see App. G). The correlation between the mean values of the ultrasound measurements at 7 and 16 weeks of age and the FCI-grade simplified as free (grade A or B) or dysplastic (grade C, D or E) are displayed in table 4.4. Only the mean values of the ultrasound measurements at 16 weeks of age on the right hip is significantly correlated with being free or dysplastic. Table 4.5 Duncan's Multiple Range Test - Means with the same letter are not significantly different Age in weeks Hip Free or dysplastic Mean value of ultrasound measurement Duncan grouping 7 Left Free A Dysplastic A 7 Right Free A Dysplastic A 16 Left Free A Dysplastic A 16 Right Free A Dysplastic B Page 24 of 36

29 5. Discussion After CHD was first described in 1937 it soon became apparent, that the disease was more common than first assumed. The need for breeding programs to eliminate CHD had to be instated. For more than 40 years the standard ventrodorsal hip-extended radiograph has been the cornerstone of every major effort to screen the population in the attempt to control the disease. It has proven efficient to a certain extent, but is by no means perfect. Since the technique relies mainly on the secondary changes that occur in connection with CHD there is a possibility of getting false-negative results in the dogs that are genetically predisposed for CHD, but have not had sufficient environmental stimuli to trigger the disease. The primary challenge in combating CHD is the fact that the disease does not necessarily develop in genetically predisposed animals. These carrier-animals, which are not subjected to enough environmental stimuli to trigger the disease, will be able to pass on their faulty genes to the next generation. It would also be greatly beneficial to evaluate the probability of developing CHD at an earlier age, than is standard at present. The final evaluation of prospective breeding stock can not be done until the dog is full grown, i.e. at age 1 to 2 years depending on breed. The PennHip method was developed based on the fact, that joint laxity is present before the secondary changes occur. It is fundamentally different from the standard ventrodorsal radiography in the respect, that it does not assign degrees of CHD, but provides a probability for later development of CHD. It has proved reliable from 4 months of age. Also it is based on objective measurements and not subjective evaluation like the standard ventrodorsal radiography. PennHip also has a lower inter- and intraobserver variance. However there are certain drawbacks. Firstly it requires certification by the PennHip Foundation. Secondly all radiographs have to be sent the USA for reading. Thirdly the technique itself requires several helpers to be present in the radiography room during the procedure. The latter is concerning because of the radiation hygiene. In the attempt to develop screening methods based on non-ionizing radiation e.g. ultrasound two different approaches were taken. Measuring angles analogous to the human method was at the time the practical part of this study was undertaken deemed inefficient (63;64). Recently it has been shown that this approach might still prove viable. (65) Page 25 of 36