BACKGROUND
diseases of the prostate are common in middle-aged to older intact dogs.
the canine prostate can be affected by benign prostatic hyperplasia , squamous metaplasia, cysts, inflammation and neoplasia.
bph, the most common prostatic disorder, is a spontaneous disease of intact male dogs that begins as glandular hyperplasia as early as 2 years of age  <cit> .
dihydrotestosterone is accepted as a key hormone in the pathogenesis of the disease, stimulating enlargement of the prostate by enhancing growth of both the stromal and glandular components  <cit> .
oestrogens, in particular the ratio of 17β-oestradiol/testosterone, are also involved in the development of bph, as is potentially prolactin  <cit>  and also other mitogenic growth factors  <cit> .
when present, clinical signs of bph include serosanguinous urethral discharge not associated with urination  <cit> , hemospermia and hematuria, tenesmus, poor semen quality and infertility, and prostatomegaly  <cit> , as well as constipation and dysuria  <cit> .
the diagnosis of bph is usually based on history, physical and/or andrologic exam and abdominal ultrasonography; and seldom on cytology by fine needle-aspiration .
palpation usually reveals a symmetrically enlarged and non-painful prostate.
the presence of prostatomegaly confirmed by ultrasonography  is frequently sufficient to reach a conclusive diagnosis of bph  <cit> .
however, bph can be difficult to differentiate from other prostatic diseases, including squamous metaplasia, prostatitis, prostatic cysts and abscesses, and prostatic neoplasia, due to the similarity of clinical and ultrasonographic findings.
under these circumstances, other methods should be used, i.e., cytology or biopsy, and more recently the seric quantification of canine prostate-specific arginine esterase   <cit> .
cpse, a serine protease similar to the human prostate-specific antigen , is the most important specific androgen-dependent protein of prostatic secretion in dog and a remarkable marker of androgenic stimulation  <cit> .
serum cpse concentrations, as determined by immunoprecipitation, were found to be significantly elevated in dogs with bph compared to normal dogs  <cit> .
cpse determination by enzyme-linked immunosorbent assay  is currently an interesting method for bph diagnosis, especially indicated to early detect dogs with a subclinical pathological condition, and opens new perspectives to the follow-up of medical treatment  <cit> .
however, there is scarce information on cpse levels in middle-aged healthy dogs.
in such animals, the determination of cpse concentration is a feasible procedure that can be of high importance for early bph diagnosis.
the aims of the present study were: a) to compare cpse levels in dogs with bph  and without bph ; and b) to compare clinical, ultrasonographic and cpse performances in the diagnosis of bph, considering the cytological marks as the reference method for this study.
methods
clinical, ultrasonographic and cytological evaluations
all the dogs  were evaluated only after an informed consent obtained from their owners.
each dog underwent clinical examination including a thorough history, physical and digital rectal examination  of the prostate, ultrasonography and cytological evaluation.
clinical manifestations compatible with bph or cystic prostatic hyperplasia  comprised constipation, ribbon-like stools, hemorrhagic urethral discharge and/or intermittent hematuria, weight loss, rear limbs pain and abnormal dre, such as palpable prostatomegaly and painful or abnormal prostate such as nodularity, indurations or asymmetry  <cit> .
prostatitis was suspected when clinical signs were more severe than bph, as caudal abdominal or pelvic pain, and a diffuse to asymmetrical enlargement and/or prostatic pain was noted at dre  <cit> .
additional procedures carried out comprised prostatic ultrasonography, prostatic massage or prostatic fna for cytological evaluation .
transabdominal ultrasonography of the prostate was performed with a philips hd <dig> ultrasound system , using a sectorial  <dig> - <dig>  mhz and linear  <dig> - <dig>  mhz transducers.
the ultrasonographic examination enabled the evaluation of the prostate size, namely length  and height in the longitudinal plan , and height  and width  in the transverse one, plus shape, position, internal echogenicity and margins.
the values for l, hl/ht and w were the average of three consecutive measurements.
prostatic volume  was determined by the formula pv  =  <dig>  × l × w ×  +  <dig> ; and the expected volume  was determined by the formula ev  =  <dig>  +   <cit> .
all variations of the normal ultrasonographic aspect were recorded .
abnormal records of ultrasonography included bph-suggestive, cystic prostatic hyperplasia -suggestive and prostatitis-suggestive ultrasonograms.
ultrasonographic findings were subdivided into simple bph, cph and prostatitis.
findings compatible with bph included symmetrical prostatic enlargement and diffusely hyperechoic parenchyma, whereas cph showed hyperechoic parenchyma background with cavitary lesions of varying architecture and distal acoustic enhancement.
prostatitis was suspected when ultrasonographic findings were more pronounced than bph, being the prostate variably hyperechoic with a more complex contour  <cit> .fig.
<dig> fine needle-aspirate from a prostate with benign hyperplasia .
cells are arranged in clusters and show a uniform morphology with round nucleus and basophilic cytoplasm
fig.
<dig> fine needle-aspirate from a normal prostate .
cells are arranged in small clusters with a uniform morphology, showing round nucleus and clear cytoplasm
for cytology, samples were collected by ultrasound-guided transabdominal fna, as previously described  <cit>  or by prostatic massage, namely when ultrasonography suggested prostatitis  <cit> .
the cells were then smeared on a slide, air-dried, stained with diff-quick and mounted with entellantm .
the presence of uniform sheets or clusters of prostatic cells with middle anysokaryosis and increased size with round and small nucleus, with a basophilic, moderate cytoplasm are the cytological criteria to determine the presence of bph  <cit> .
these clusters could be in a high quantity but their cells appeared as normal epithelial prostatic ones.
the cytology of prostatitis is determined by the presence of a large number of neutrophils associated with a variable numbers of macrophages .
bacteria could be present, both freely or intracytoplasmatically .
finally, squamous metaplasia is determined by the presence of prostatic cells with squamous epithelial characteristics such as large and clear to basophilic cytoplasm with small dense nucleus  <cit> .
groups of animals
based on cytological results, two groups of adult intact male dogs were formed, i.e., a study group  and a control group .table  <dig> records from clinical examination, ultrasonography and cytology in  <dig> dogs  diagnosed with benign prostatic hyperplasia  by cytological examination of prostatic aspirates
cph cystic prostatic hyperplasia, dre digital rectal examination
cph cystic prostatic hyperplasia, dre digital rectal examination
group a included eight mongrels and  <dig> other dogs from  <dig> defined breeds, i.e., labrador retriever , boxer , estrela mountain dog , golden retriever , beagle , brittany , chow-chow , cocker spaniel , dalmatian , pekingese , portuguese sheepdog , rottweiler  and siberian husky .
weight ranged from  <dig> to 59 kg, with a median of  <dig>  kg .
age ranged from  <dig> to 15 years, with a median of  <dig>  years .
group c included eight mongrels and  <dig> other dogs from  <dig> defined breeds, i.e., beagle , brittany , german shepherd , golden retriever , cocker spaniel , drever , estrela mountain dog , french bulldog , german spitz klein , german spitz mittel , labrador retriever , miniature pinscher , portuguese pointer , portuguese water dog , st. bernard  and transmontano mastiff .
weight ranged from  <dig> to 70 kg, with a median of  <dig>  kg .
age ranged from  <dig> to 12 years, with a median of  <dig>  years .
canine prostate-specific arginine esterase
blood was collected from all the dogs, left to clot for 4 h at room temperature and the collected serum kept frozen at −25 °c until use.
in order to measure plasma cpse concentrations, an enzyme-linked immunosorbent assay  was used strictly following the manufacturer’s instructions .
briefly, dilutions of the elisa stock solution were done to obtain the different calibrator solutions  and to define the standard curve.
all the plasma samples were diluted at 1: <dig> and incubated at 37 °c for 1 h before being assayed.
the horseradish peroxidase conjugate were placed into each well after washing cycle and incubated again at 37 °c for 1 h. after another washing cycle, tetramethylbenzidine was added for 10 min and the reaction then stopped.
the optical density  of each sample was read at 450 nm and cpse plasma concentrations calculated based on the standard curve plotted with calibrator concentrations.
a cpse value of 61 ng/ml was set as the cut-off for bph , according to the manufacturer’s instructions.
data analysis
the agreement beyond chance between binomial results  of cpse and those of the clinical, ultrasonographic and cytological evaluations for bph was measured with cohen’s kappa coefficient .
sensitivity , specificity , positive likelihood ratio  and negative likelihood ratio  were calculated for cpse binomial results taking cytology as the reference method.
exact binomial 95% confidence intervals  were established for proportions.
differences in the cpse levels between groups a and c were assessed by means of the mann-whitney u test.
the association between cpse levels and other quantitative variables  was measured by spearman’s rank correlation coefficient r  <cit> .
analyses were performed with spss  <dig>  software for windows, with a p value <  <dig>  as statistically significant.
RESULTS
cpse levels between groups
the levels of cpse  were significantly different  between group a  and group c .
significant  correlations were found between age and cpse level  and cpse and pv ; but not between cpse and ev .fig.
<dig> canine prostate-specific arginine esterase  levels in  <dig> dogs  with benign prostatic hyperplasia , as diagnosed by cytology; and in  <dig> dogs  without bph .
the dotted line represents the cut-off value for cpse
clinical, ultrasonographic and cytological records
in group a , all  <dig> dogs evidenced bph confirmed by cytology; moreover, eight  dogs showed prostatitis concomitant with bph .
in group c , six dogs had prostatitis and two other had squamous metaplasia .
comparison between cpse levels, clinical, ultrasonographic and cytological evaluations
the k values from comparisons between binomial cpse levels and clinic evaluation, ultrasonography and cytology were  <dig> ,  <dig>  and  <dig> , respectively .
a k value above  <dig>  represents an almost perfect agreement beyond chance.table  <dig> comparison between the binomial results of canine prostate-specific arginine esterase  and those of clinical evaluation, ultrasonography and cytology for the diagnosis of benign prostatic hyperplasia  in dogs
cpsea, b, c
a: k =  <dig> ; b: k =  <dig> ; c: k =  <dig> ; +ve: positive; −ve: negative
three out of the  <dig> dogs of group c had a positive cpse result.
two of those three dogs had prostatitis and cpse values of  <dig>  and  <dig>  ng/ml; the other animal had squamous metaplasia and a cpse value of  <dig>  ng/ml.
one dog of group a had a cpse level of  <dig>  ng/ml.
sensitivity of the cpse binomial results  was  <dig> % , specificity  <dig> % , positive likelihood ratio  <dig>   and negative likelihood ratio  <dig>  .
a positive likelihood ratio of  <dig>  reveals a moderate to large and often conclusive increase in the likelihood of bph if the cpse result is positive.
on the other side, a negative likelihood ratio of  <dig>  stands for a large and often conclusive decrease in the likelihood of bph if the cpse result is negative.
discussion
prostatic diseases are frequent age-related conditions in intact dogs.
the importance of applying a systematic diagnostic protocol that allows an accurate identification of prostatic disorders should be emphasized, as this provides the chance of an early control of the disorder without development of chronic disease, which is hard to manage and causes deterioration of fertility  <cit> .
several diagnostic methods can be used to evaluate the prostate, from physical examination  to ultrasonography, assessment of ejaculate’s third fraction, prostatic massage, prostatic fna and biopsy  <cit> .
data obtained from clinical examination are important as starting points for further evaluation of the prostate.
nevertheless, clinical signs of prostatic disease can be transitory and unspecific, precluding a desirably early and conclusive diagnosis.
assessment of the ejaculate’s third fraction could have been an adequate criterion to evaluate prostatic disease.
however, taking into account that males included in the study were not breed dogs, semen collection was not attempted.
prostatic ultrasonography is an excellent method in the evaluation of the position, size, contours, and parenchymal echogenicity, as well as in the evaluation of the prostatic urethra.
jointly, clinical evaluation of the prostate and ultrasonography could allow a presumptive diagnosis of the prostatic condition in a reasonable number of cases, but sensitivity would not be optimal .
other methods should then be carried out, such as the prostatic massage and/or the invasive fna and biopsy  <cit> .
ultrasound-guided transabdominal fna was used in this study as the preferred method to obtain prostatic tissue and fluid, and then as the gold standard to which the other methods were compared, considering the very high agreement between fna and histopathological diagnosis  <cit>  and also the availability of this technique.
besides, the thin monolayer obtained with cytological smears often allows better assessment of individual cytomorphology, as well as improved detection of etiologic agents  <cit> .
prostatic biopsy is only considered in cases where less invasive diagnostic tests do not render a conclusive diagnosis  <cit> .
under the above-mentioned circumstances, when it comes to obtain an accurate diagnosis prior to initiating a treatment, several tests can be carried out, taking always into account the benefits, costs and risks of each procedure.
also considering the fact that there is still room for improvement, novel molecular tools have been proposed to early detect prostatic diseases such as the cpse elisa kit, which allows determination of cpse levels in an expeditious manner.
in the present study, cpse levels were determined after the formation of two defined groups, i.e., a study and a control, in which animals were cytologically confirmed as having bph or not having-bph, respectively.
cpse serum values were significantly higher in bph than in non-bph dogs, which indicate an increased production and secretion of this marker by prostatic epithelial cells in animals with bph, and further confirm the diagnostic value of the test.
furthermore, seven dogs in group a  had at least one clinical sign but no changes at dre, while seven other showed no clinical signs.
considering that these dogs were older than 5 years old, the present study highlights the importance of systematic cpse quantification in middle-aged dogs even when apparently healthy.
an early bph diagnosis in these dogs is always desirable considering the impact of the disorder in fertility  <cit> .
cpse determination is furthermore useful when ultrasonography is not available or for diagnosis in large and obese dogs in which dre could be impossible, and also of great utility in the follow-up of bph medical treatment.
cpse determination does not preclude the attainment of other complementary tests used to exclude the coexistence of other prostatic diseases, such as prostatitis, squamous metaplasia or neoplasia, which are especially recommended when clinical examination and/or ultrasonography are suggestive of disorders other than bph.
cpse levels were moderately correlated with prostatic volume , which could be explained by the elevated secretion of cpse in enlarged hyperplasic prostates.
there was no correlation between cpse and the expected volume  of the prostate, as the latter adjusts prostate size to the weight of dogs.
another important finding is the fact that, although there were significant differences between median weights of the dogs in the two groups, no statistically significant correlation was detected between weight and cpse levels, a fact which indicates reliability of the test for large breeds.
in group a , seven out of  <dig> dogs with bph – i.e., with high cpse values – also presented prostatitis.
the association of various prostatic disorders is not uncommon; in fact, diseased prostate glands are commonly affected by multiple and even co-existing pathological conditions  <cit> .
cph could predispose prostate to inflammation by providing a favorable medium for bacterial growth or by interference with normal defense mechanisms  <cit> .
the hypothesis that inflammation emerges as the major contributor for the development of bph has been suggested in men  <cit> .
on the other hand, in group c , six out of  <dig> dogs presented prostatitis, with two of them showing cpse increased values, which could indicate the need of following up these animals.
previous studies have found no difference between serum concentrations of cpse in dogs with bph and in those with bacterial prostatitis and carcinoma, which was attributed to the existence of concurrent bph  <cit> .
however, the method for cpse determination was different from the current one.
in the present study, this could be excluded considering that cytological analysis ruled out bph in group c. the remaining dogs of group c with prostatitis showed low cpse values, like the dogs without bph in the same group.
further studies with large numbers of dogs should contribute to clarify these findings.
CONCLUSIONS
results of the present study revealed a very high agreement  between cpse test and other diagnostic tools for bph diagnosis.
previous studies have suggested cytological evaluation as one of the methods with higher accuracy in the diagnosis of prostatic diseases  <cit> , which was also observed in the present study, with the highest agreement found between the cpse test and cytological diagnosis.
the cpse test can very accurately differentiate between dogs with and without bph and may be considered as an alternative or complementary tool to conventional methods for bph diagnosis.
finally, its systematic use can increase the likelihood of early diagnosing bph in apparently healthy middle-aged dogs.
additional files
additional file 1: : signalment, clinical diagnosis, ultrasonographic features, cytological diagnosis, prostatic dimensions and cpse levels in  <dig> dogs with bph as determined by cytology.
additional file 2: : signalment, clinical diagnosis, ultrasonographic features, cytological diagnosis, prostatic dimensions and cpse levels in  <dig> dogs without evidence of bph by cytology.
abbreviations
bphbenign prostatic hyperplasia
ciconfidence interval
cphcystic prostatic hyperplasia
cpsecanine prostate-specific arginine esterase
elisaenzyme-linked immunosorbent assay
evexpected volume
fnafine needle-aspiration
iqrinterquartile range
pvprostatic volume
rderectal digital examination

 diseases of the prostate are common in middle-aged to older intact dogs. the canine prostate can be affected by benign prostatic hyperplasia, squamous metaplasia, cysts, inflammation and neoplasia. bph, the most common prostatic disorder, is a spontaneous disease of intact male dogs that begins as glandular hyperplasia as early as 2 years of age. dihydrotestosterone is accepted as a key hormone in the pathogenesis of the disease, stimulating enlargement of the prostate by enhancing growth of both the stromal and glandular components. oestrogens, in particular the ratio of 17β-oestradiol/testosterone, are also involved in the development of bph, as is potentially prolactin and also other mitogenic growth factors. when present, clinical signs of bph include serosanguinous urethral discharge not associated with urination, hemospermia and hematuria, tenesmus, poor semen quality and infertility, and prostatomegaly, as well as constipation and dysuria. the diagnosis of bph is usually based on history, physical and/or andrologic exam and abdominal ultrasonography; and seldom on cytology by fine needle-aspiration. palpation usually reveals a symmetrically enlarged and non-painful prostate. the presence of prostatomegaly confirmed by ultrasonography is frequently sufficient to reach a conclusive diagnosis of bph. however, bph can be difficult to differentiate from other prostatic diseases, including squamous metaplasia, prostatitis, prostatic cysts and abscesses, and prostatic neoplasia, due to the similarity of clinical and ultrasonographic findings. under these circumstances, other methods should be used, i.e., cytology or biopsy, and more recently the seric quantification of canine prostate-specific arginine esterase. cpse, a serine protease similar to the human prostate-specific antigen, is the most important specific androgen-dependent protein of prostatic secretion in dog and a remarkable marker of androgenic stimulation. serum cpse concentrations, as determined by immunoprecipitation, were found to be significantly elevated in dogs with bph compared to normal dogs. cpse determination by enzyme-linked immunosorbent assay is currently an interesting method for bph diagnosis, especially indicated to early detect dogs with a subclinical pathological condition, and opens new perspectives to the follow-up of medical treatment. however, there is scarce information on cpse levels in middle-aged healthy dogs. in such animals, the determination of cpse concentration is a feasible procedure that can be of high importance for early bph diagnosis. the aims of the present study were: a) to compare cpse levels in dogs with bph and without bph; and b) to compare clinical, ultrasonographic and cpse performances in the diagnosis of bph, considering the cytological marks as the reference method for this study. methods clinical, ultrasonographic and cytological evaluations all the dogs were evaluated only after an informed consent obtained from their owners. each dog underwent clinical examination including a thorough history, physical and digital rectal examination of the prostate, ultrasonography and cytological evaluation. clinical manifestations compatible with bph or cystic prostatic hyperplasia comprised constipation, ribbon-like stools, hemorrhagic urethral discharge and/or intermittent hematuria, weight loss, rear limbs pain and abnormal dre, such as palpable prostatomegaly and painful or abnormal prostate such as nodularity, indurations or asymmetry. prostatitis was suspected when clinical signs were more severe than bph, as caudal abdominal or pelvic pain, and a diffuse to asymmetrical enlargement and/or prostatic pain was noted at dre. additional procedures carried out comprised prostatic ultrasonography, prostatic massage or prostatic fna for cytological evaluation. transabdominal ultrasonography of the prostate was performed with a philips hd ultrasound system, using a sectorial -  mhz and linear -  mhz transducers. the ultrasonographic examination enabled the evaluation of the prostate size, namely length and height in the longitudinal plan, and height and width in the transverse one, plus shape, position, internal echogenicity and margins. the values for l, hl/ht and w were the average of three consecutive measurements. prostatic volume was determined by the formula pv  =   × l × w ×  + ; and the expected volume was determined by the formula ev  =   + . all variations of the normal ultrasonographic aspect were recorded. abnormal records of ultrasonography included bph-suggestive, cystic prostatic hyperplasia -suggestive and prostatitis-suggestive ultrasonograms. ultrasonographic findings were subdivided into simple bph, cph and prostatitis. findings compatible with bph included symmetrical prostatic enlargement and diffusely hyperechoic parenchyma, whereas cph showed hyperechoic parenchyma background with cavitary lesions of varying architecture and distal acoustic enhancement. prostatitis was suspected when ultrasonographic findings were more pronounced than bph, being the prostate variably hyperechoic with a more complex contour.fig. fine needle-aspirate from a prostate with benign hyperplasia. cells are arranged in clusters and show a uniform morphology with round nucleus and basophilic cytoplasm fig. fine needle-aspirate from a normal prostate. cells are arranged in small clusters with a uniform morphology, showing round nucleus and clear cytoplasm for cytology, samples were collected by ultrasound-guided transabdominal fna, as previously described or by prostatic massage, namely when ultrasonography suggested prostatitis. the cells were then smeared on a slide, air-dried, stained with diff-quick and mounted with entellantm. the presence of uniform sheets or clusters of prostatic cells with middle anysokaryosis and increased size with round and small nucleus, with a basophilic, moderate cytoplasm are the cytological criteria to determine the presence of bph. these clusters could be in a high quantity but their cells appeared as normal epithelial prostatic ones. the cytology of prostatitis is determined by the presence of a large number of neutrophils associated with a variable numbers of macrophages. bacteria could be present, both freely or intracytoplasmatically. finally, squamous metaplasia is determined by the presence of prostatic cells with squamous epithelial characteristics such as large and clear to basophilic cytoplasm with small dense nucleus. groups of animals based on cytological results, two groups of adult intact male dogs were formed, i.e., a study group and a control group.table records from clinical examination, ultrasonography and cytology in dogs diagnosed with benign prostatic hyperplasia by cytological examination of prostatic aspirates cph cystic prostatic hyperplasia, dre digital rectal examination cph cystic prostatic hyperplasia, dre digital rectal examination group a included eight mongrels and other dogs from defined breeds, i.e., labrador retriever, boxer, estrela mountain dog, golden retriever, beagle, brittany, chow-chow, cocker spaniel, dalmatian, pekingese, portuguese sheepdog, rottweiler and siberian husky. weight ranged from to 59 kg, with a median of  kg. age ranged from to 15 years, with a median of  years. group c included eight mongrels and other dogs from defined breeds, i.e., beagle, brittany, german shepherd, golden retriever, cocker spaniel, drever, estrela mountain dog, french bulldog, german spitz klein, german spitz mittel, labrador retriever, miniature pinscher, portuguese pointer, portuguese water dog, st. bernard and transmontano mastiff. weight ranged from to 70 kg, with a median of  kg. age ranged from to 12 years, with a median of  years. canine prostate-specific arginine esterase blood was collected from all the dogs, left to clot for 4 h at room temperature and the collected serum kept frozen at −25 °c until use. in order to measure plasma cpse concentrations, an enzyme-linked immunosorbent assay was used strictly following the manufacturer’s instructions. briefly, dilutions of the elisa stock solution were done to obtain the different calibrator solutions and to define the standard curve. all the plasma samples were diluted at 1: and incubated at 37 °c for 1 h before being assayed. the horseradish peroxidase conjugate were placed into each well after washing cycle and incubated again at 37 °c for 1 h. after another washing cycle, tetramethylbenzidine was added for 10 min and the reaction then stopped. the optical density of each sample was read at 450 nm and cpse plasma concentrations calculated based on the standard curve plotted with calibrator concentrations. a cpse value of 61 ng/ml was set as the cut-off for bph, according to the manufacturer’s instructions. data analysis the agreement beyond chance between binomial results of cpse and those of the clinical, ultrasonographic and cytological evaluations for bph was measured with cohen’s kappa coefficient. sensitivity, specificity, positive likelihood ratio and negative likelihood ratio were calculated for cpse binomial results taking cytology as the reference method. exact binomial 95% confidence intervals were established for proportions. differences in the cpse levels between groups a and c were assessed by means of the mann-whitney u test. the association between cpse levels and other quantitative variables was measured by spearman’s rank correlation coefficient r. analyses were performed with spss software for windows, with a p value <  as statistically significant.  cpse levels between groups the levels of cpse were significantly different between group a and group c. significant correlations were found between age and cpse level and cpse and pv; but not between cpse and ev.fig. canine prostate-specific arginine esterase levels in dogs with benign prostatic hyperplasia, as diagnosed by cytology; and in dogs without bph. the dotted line represents the cut-off value for cpse clinical, ultrasonographic and cytological records in group a, all dogs evidenced bph confirmed by cytology; moreover, eight dogs showed prostatitis concomitant with bph. in group c, six dogs had prostatitis and two other had squamous metaplasia. comparison between cpse levels, clinical, ultrasonographic and cytological evaluations the k values from comparisons between binomial cpse levels and clinic evaluation, ultrasonography and cytology were, and, respectively. a k value above represents an almost perfect agreement beyond chance.table comparison between the binomial results of canine prostate-specific arginine esterase and those of clinical evaluation, ultrasonography and cytology for the diagnosis of benign prostatic hyperplasia in dogs cpsea, b, c a: k = ; b: k = ; c: k = ; +ve: positive; −ve: negative three out of the dogs of group c had a positive cpse result. two of those three dogs had prostatitis and cpse values of and  ng/ml; the other animal had squamous metaplasia and a cpse value of  ng/ml. one dog of group a had a cpse level of  ng/ml. sensitivity of the cpse binomial results was %, specificity %, positive likelihood ratio and negative likelihood ratio. a positive likelihood ratio of reveals a moderate to large and often conclusive increase in the likelihood of bph if the cpse result is positive. on the other side, a negative likelihood ratio of stands for a large and often conclusive decrease in the likelihood of bph if the cpse result is negative. discussion prostatic diseases are frequent age-related conditions in intact dogs. the importance of applying a systematic diagnostic protocol that allows an accurate identification of prostatic disorders should be emphasized, as this provides the chance of an early control of the disorder without development of chronic disease, which is hard to manage and causes deterioration of fertility. several diagnostic methods can be used to evaluate the prostate, from physical examination to ultrasonography, assessment of ejaculate’s third fraction, prostatic massage, prostatic fna and biopsy. data obtained from clinical examination are important as starting points for further evaluation of the prostate. nevertheless, clinical signs of prostatic disease can be transitory and unspecific, precluding a desirably early and conclusive diagnosis. assessment of the ejaculate’s third fraction could have been an adequate criterion to evaluate prostatic disease. however, taking into account that males included in the study were not breed dogs, semen collection was not attempted. prostatic ultrasonography is an excellent method in the evaluation of the position, size, contours, and parenchymal echogenicity, as well as in the evaluation of the prostatic urethra. jointly, clinical evaluation of the prostate and ultrasonography could allow a presumptive diagnosis of the prostatic condition in a reasonable number of cases, but sensitivity would not be optimal. other methods should then be carried out, such as the prostatic massage and/or the invasive fna and biopsy. ultrasound-guided transabdominal fna was used in this study as the preferred method to obtain prostatic tissue and fluid, and then as the gold standard to which the other methods were compared, considering the very high agreement between fna and histopathological diagnosis and also the availability of this technique. besides, the thin monolayer obtained with cytological smears often allows better assessment of individual cytomorphology, as well as improved detection of etiologic agents. prostatic biopsy is only considered in cases where less invasive diagnostic tests do not render a conclusive diagnosis. under the above-mentioned circumstances, when it comes to obtain an accurate diagnosis prior to initiating a treatment, several tests can be carried out, taking always into account the benefits, costs and risks of each procedure. also considering the fact that there is still room for improvement, novel molecular tools have been proposed to early detect prostatic diseases such as the cpse elisa kit, which allows determination of cpse levels in an expeditious manner. in the present study, cpse levels were determined after the formation of two defined groups, i.e., a study and a control, in which animals were cytologically confirmed as having bph or not having-bph, respectively. cpse serum values were significantly higher in bph than in non-bph dogs, which indicate an increased production and secretion of this marker by prostatic epithelial cells in animals with bph, and further confirm the diagnostic value of the test. furthermore, seven dogs in group a had at least one clinical sign but no changes at dre, while seven other showed no clinical signs. considering that these dogs were older than 5 years old, the present study highlights the importance of systematic cpse quantification in middle-aged dogs even when apparently healthy. an early bph diagnosis in these dogs is always desirable considering the impact of the disorder in fertility. cpse determination is furthermore useful when ultrasonography is not available or for diagnosis in large and obese dogs in which dre could be impossible, and also of great utility in the follow-up of bph medical treatment. cpse determination does not preclude the attainment of other complementary tests used to exclude the coexistence of other prostatic diseases, such as prostatitis, squamous metaplasia or neoplasia, which are especially recommended when clinical examination and/or ultrasonography are suggestive of disorders other than bph. cpse levels were moderately correlated with prostatic volume, which could be explained by the elevated secretion of cpse in enlarged hyperplasic prostates. there was no correlation between cpse and the expected volume of the prostate, as the latter adjusts prostate size to the weight of dogs. another important finding is the fact that, although there were significant differences between median weights of the dogs in the two groups, no statistically significant correlation was detected between weight and cpse levels, a fact which indicates reliability of the test for large breeds. in group a, seven out of dogs with bph – i.e., with high cpse values – also presented prostatitis. the association of various prostatic disorders is not uncommon; in fact, diseased prostate glands are commonly affected by multiple and even co-existing pathological conditions. cph could predispose prostate to inflammation by providing a favorable medium for bacterial growth or by interference with normal defense mechanisms. the hypothesis that inflammation emerges as the major contributor for the development of bph has been suggested in men. on the other hand, in group c, six out of dogs presented prostatitis, with two of them showing cpse increased values, which could indicate the need of following up these animals. previous studies have found no difference between serum concentrations of cpse in dogs with bph and in those with bacterial prostatitis and carcinoma, which was attributed to the existence of concurrent bph. however, the method for cpse determination was different from the current one. in the present study, this could be excluded considering that cytological analysis ruled out bph in group c. the remaining dogs of group c with prostatitis showed low cpse values, like the dogs without bph in the same group. further studies with large numbers of dogs should contribute to clarify these findings.  results of the present study revealed a very high agreement between cpse test and other diagnostic tools for bph diagnosis. previous studies have suggested cytological evaluation as one of the methods with higher accuracy in the diagnosis of prostatic diseases, which was also observed in the present study, with the highest agreement found between the cpse test and cytological diagnosis. the cpse test can very accurately differentiate between dogs with and without bph and may be considered as an alternative or complementary tool to conventional methods for bph diagnosis. finally, its systematic use can increase the likelihood of early diagnosing bph in apparently healthy middle-aged dogs. additional files additional file 1:: signalment, clinical diagnosis, ultrasonographic features, cytological diagnosis, prostatic dimensions and cpse levels in dogs with bph as determined by cytology. additional file 2:: signalment, clinical diagnosis, ultrasonographic features, cytological diagnosis, prostatic dimensions and cpse levels in dogs without evidence of bph by cytology. abbreviations bphbenign prostatic hyperplasia ciconfidence interval cphcystic prostatic hyperplasia cpsecanine prostate-specific arginine esterase elisaenzyme-linked immunosorbent assay evexpected volume fnafine needle-aspiration iqrinterquartile range pvprostatic volume rderectal digital examination 
the aim of the present study was to verify cpse levels in negative controls and hyperplasic dogs, considering cytological findings as the reference method and taking into account the fact that controls were middle-aged intact dogs, contrarily to previous studies carried out with very young control dogs. significant differences of median cpse levels were found between controls and hyperplasic dogs; and significant positive correlations were found between median cpse levels and age or prostatic volume. the agreement between methods was found to be very high, notably between cpse levels and cytological results. benign prostatic hyperplasia is the most common canine prostatic disorder. taking into account the non-specific character of clinical and ultrasonographic findings, a new diagnostic approach has recently been proposed based on the augmentation of blood canine prostate-specific arginine esterase in hyperplasic dogs. considering the results all together, measurement of cpse is confirmed as a useful and accurate method and should be considered as an alternative or complementary tool to conventional methods for the diagnosis of bph in middle-aged dogs. although most or even all intact male dogs may develop bph by 5– years of age, many show no clinical signs.
 benign prostatic hyperplasia is the most common canine prostatic disorder. although most or even all intact male dogs may develop bph by 5– years of age, many show no clinical signs. taking into account the non-specific character of clinical and ultrasonographic findings, a new diagnostic approach has recently been proposed based on the augmentation of blood canine prostate-specific arginine esterase in hyperplasic dogs. the aim of the present study was to verify cpse levels in negative controls and hyperplasic dogs, considering cytological findings as the reference method and taking into account the fact that controls were middle-aged intact dogs, contrarily to previous studies carried out with very young control dogs.  significant differences of median cpse levels were found between controls and hyperplasic dogs; and significant positive correlations were found between median cpse levels and age or prostatic volume. sensitivity, specificity, positive and negative likelihood ratios put into evidence the good performance of the test. the agreement between methods was found to be very high, notably between cpse levels and cytological results.  considering the results all together, measurement of cpse is confirmed as a useful and accurate method and should be considered as an alternative or complementary tool to conventional methods for the diagnosis of bph in middle-aged dogs. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords prostatebenign prostate hyperplasiabphcanine prostate-specific arginine esterasecpsebiomarkerhttp://dx.doi.org/http://dx.doi.org/ /501100001871fundação para a ciência e a tecnologiaprojects pest-oe/agr/ui0772/2011issue-copyright-statement© the author 2017 