C-reactive Protein in the Differentiation of Pyometra From Cystic Endometrial Hyperplasia/Mucometra in Dogs
Hematological parameters, plasma C-reactive protein (CRP), and tumor necrosis factor αwere analyzed in 64 dogs with a presumptive diagnosis of pyometra. Final diagnosis (i.e., pyometra or cystic endometrial hyperplasia [CEH]) was determined by histopathology. As a single test, the percentage of band neutrophils had the highest sensitivity in the prediction of pyometra (sensitivity, 94%). The combination of percentage of bands and CRP had the highest sensitivity (97.7%; specificity, 75%) in predicting the presence of pyometra. The most common clinical signs noted in the study were vaginal discharge, polyuria, polydipsia, lethargy, and gastrointestinal signs. A combination of three or more of these clinical signs was significantly associated with pyometra.
Introduction
Canine pyometra is considered part of the cystic endometrial hyperplasia (CEH)-pyometra complex, a concept introduced by Dow in 1957.1 Clinically, however, there are many differences between CEH and pyometra. In most cases of pyometra, bacterial growth in the uterus and the subsequent immune response affect the dog systemically.2–8 The most common bacteria isolated from the uterus is Escherichia coli (E. coli).24 Like other gram-negative bacteria, E. coli may release endotoxin during vigorous growth or upon their death.9 Endotoxemia is believed to be responsible for many of the clinical signs associated with pyometra.7810 If rupture of the infected uterus occurs, bacterial peritonitis and/or sepsis may result, with a guarded to poor prognosis.11
In contrast, CEH is not associated with bacterial infection of the uterus, and systemic effects have not been demonstrated.212 Recently, after uterine morphometric and hormone receptor analysis, it was suggested that the CEH-pyometra complex should be divided into two separate entities (i.e., pyometra and CEH) and that both could arise de novo.1213 However, it is generally accepted that CEH renders the uterus susceptible to bacterial infection and that CEH often precedes pyometra.2–8
Despite the differences in their pathology, CEH and pyometra can be difficult to differentiate clinically.212 Fluid can accumulate in the uterus in both diseases, namely mucus in cases of CEH and purulent material in cases of pyometra.212 When a fluid-filled uterus is demonstrated by diagnostic imaging, the clinician may feel obligated to perform an immediate ovariohysterectomy to avoid the risk of uterine rupture. Parameters that differentiate pyometra from CEH prior to ovariohysterectomy would be very helpful in making a decision as to the most appropriate therapeutic course to pursue in these cases.
The purpose of this study was to determine criteria that clinically differentiate pyometra from CEH. Parameters evaluated included history, physical examination findings, hematological values, clinical biochemical results, and two markers of inflammation, C-reactive protein (CRP) and tumor necrosis factor alpha (TNFα). The latter two parameters were selected based on their use as inflammatory markers in humans.14–16
Materials and Methods
Case Selection
This study was approved by the board for ethical use of animals in research, Djurforsoksetiska Namnden, Tierp, Sweden. Client-owned dogs that were presented to the Department of Small Animal Clinical Sciences at the Swedish University of Agricultural Science (SLU), Uppsala, Sweden, with the presumptive diagnosis of pyometra and subsequently treated by ovariohysterectomy, were included in the study after informed owner consent. The presumptive diagnosis was based on signalment, history, clinical signs, and diagnostic imaging (i.e., demonstration of an enlarged, fluid-filled uterus on radiology or ultrasonography). The diagnosis of pyometra or CEH was confirmed by histopathological examination of the uterus in each case.
Data Collection
Prior to admission, the owners completed a questionnaire reporting the duration and presence of the following clinical signs: vaginal discharge, inappetence or vomiting, lethargy, polyuria, and polydipsia. The owners were also asked to record any other observed signs. At admission, the following physical parameters were recorded: body temperature, heart rate, respiratory rate, mucous membrane color, capillary refill time, detection of pain upon abdominal palpation, hydration status, and general attitude of the animal. Blood was collected into two ethylenediamine triacetate (EDTA) tubes and into one tube with no additive. Blood samples were placed on ice and transported within 30 minutes to the Department of Clinical Chemistry, SLU, Uppsala, Sweden, where plasma and serum were immediately separated. Immediately after separation, the plasma from one of the EDTA tubes was stored in two aliquots at −70°C (−94°F).
The following hematological and serum biochemical tests were determined: hematocrit (HCT), hemoglobin, red blood cell count (RBC) and white blood cell count (WBC), WBC differential count (including absolute counts and percentage of band neutrophils), nucleated RBCs, erythrocyte morphology, erythrocyte mean volume, erythrocyte hemoglobin content, platelet count, alanine aminotransferase, albumin, alkaline phosphatase (ALP), blood urea nitrogen, cholesterol, glucose, total protein, and the electrolytes sodium, potassium, chloride, and calcium.
Healthy, adult, intact bitches and a group of healthy bitches that had previously been treated for pyometra were used as controls. A history questionnaire was completed for all control bitches, ensuring that the dog was healthy for at least 2 months prior to examination. Physical examinations were performed by one of the authors (Bergstrom), and blood samples were obtained using the same procedures as for the diseased dogs. Only bitches with normal history, physical examination, hematology results, and serum biochemical tests were enrolled as control dogs.
Plasma samples from diseased and control dogs were transported to Washington State University, Pullman, Washington, on dry ice with a transport time of <24 hours. After confirmation that the samples were still frozen after transport, they were stored at −70°C (−94°F) until analysis. C-reactive protein was determined using a commercially available canine sandwich enzyme-linked immunosorbent assay (ELISA).a
Plasma TNFαconcentrations were determined using a sandwich ELISA.b Experimental ELISA assays using this antibody in dog plasma have shown a detection limit of 15 pg/mL, which is 1/60 of the levels achieved after sublethal doses of endotoxin.17 This ELISA test has shown good correlation with the Walter and Eliza Hall Institute of Medical Research bioassay, and it is specific for biologically active TNFαin humans.18
Pathological Examinations
Following ovariohysterectomy, the whole uterus and ovaries were fixed in 10% neutral buffered formalin. The gross appearance of the uterus was documented, and the presence of corpora lutea in the ovaries was noted. Representative sections from each uterine horn were embedded in paraffin and stained with hematoxylin and eosin.
The histopathological diagnosis of pyometra was based on fulfillment of the criteria for chronic purulent metritis, acute purulent metritis, or purulent endometritis, as proposed by Boerresen.5 Chronic purulent metritis was characterized by a macroscopically distended uterus with an irregular endometrial surface. Microscopically, varying numbers of neutrophils, plasma cells, and lymphocytes were observed in the mucosa. These cell types were also present to a lesser extent in the myometrium. The endometrial epithelium was hyperplastic and cystic with columnar cells, indicating progesterone influence. Cysts often contained large amounts of neutrophils. In some cases, a varying degree of fibrosis and/or erosions in the mucosa was seen. In purulent endometritis, observed inflammatory cells were mostly neutrophils, and cellular infiltration was limited to the endometrium. In acute purulent metritis, inflammatory changes were also present in deeper layers of the uterine wall but without chronic changes such as fibrosis. A diagnosis of CEH was based on the presence of hyperplasia of the endometrium, in combination with endometrial cysts (of different sizes), and an absence of inflammatory changes. A diagnosis of mucometra was based on gross or microscopical evidence of mucus or serous fluid in the uterine lumen in combination with a hyperplastic (and cystic) or atrophic endometrium.
Statistical Analysis
Descriptive statistics (mean and standard deviation) and two-sample unequal variance Student’s t-test with two-tailed distribution were calculated using a commercially available statistical program.c Binary criteria were compared with Fisher’s exact test. Multiple logistic regression analysis using the SAS systemd was used to evaluate the relationship between diagnosis and blood parameters, as well as the sensitivity and specificity of the resulting model to differentiate between pyometra and CEH.19 A P value <0.05 was considered significant.
Results
A total of 64 dogs with a presumptive diagnosis of pyometra were included in the study. Of these, 54 dogs were histopathologically diagnosed with pyometra. Ten dogs were diagnosed as having CEH, with (n=4) or without (n=6) mucometra. None of the dogs were pregnant. The mean age of dogs with pyometra (8.4 years; range, 1 to 12 years) was not significantly different (P=0.09) from the mean age of dogs with CEH (6.6 years; range, 1 to 10 years). The median age of both groups was 8 years. The 54 dogs with pyometra were represented by 32 different breeds. The 10 dogs with CEH were represented by nine different breeds.
Results of selected blood parameters from dogs with pyometra, with CEH, and from healthy control dogs, are presented in Table 1. The hematological and serum biochemical tests performed were based on previously demonstrated differences in dogs with pyometra versus CEH.2568 Multiple logistic regression analysis revealed that the percentage of band neutrophils was the single parameter with the highest sensitivity in differentiating pyometra from CEH (sensitivity, 94.2%; specificity, 70.0%). The most sensitive overall predictor of pyometra, however, was the combination of elevations in percentage of bands and CRP (sensitivity, 97.7%; specificity, 75%). The combination of parameters that had the highest specificity for differentiating pyometra from CEH was the percentage of bands and elevated ALP results (sensitivity, 96.1%; specificity, 77.8%). Combinations of more than two parameters did not increase the sensitivity or specificity for differentiating pyometra from CEH.
The accompanying Figure presents a contour plot of the 95% and 80% estimated probability of a dog having pyometra, in contrast to CEH, given the percentage of bands and the CRP concentration. This contour plot was generated illustrating the formula:
\(Estimated\ Probability\ of\ Pyometra\ =\ natural\ exponential\ ({-}2.3110\ +\ 0.2643\ {\times}\ percent\ bands\ +\ 0.0202\ {\times}\ CRP)\ {\div}\ 1\ +\ natural\ exponential\ ({-}2.3110\ +\ 0.2643\ {\times}\ percent\ bands\ +\ 0.0202\ {\times}\ CRP)\)The clinical signs most consistently observed were vaginal discharge, polyuria, polydipsia, lethargy, and gastrointestinal signs (e.g., vomiting, inappetence) [Table 2]. The subjective and objective physical examination abnormalities in dogs with pyometra and CEH are presented in Table 3.
Discussion
In the study reported here, the most useful laboratory parameters for the differentiation of canine pyometra from CEH were the percentage of band neutrophils, ALP levels, and CRP concentrations. The first two abnormalities were consistent with previously reported results in cases of pyometra (as compared to other uterine diseases).2 In this study, the WBC counts of dogs with pyometra were significantly higher than those of dogs with CEH; however, multiple logistic regression analysis revealed that the percentage of bands in the WBC was a more sensitive indicator of pyometra.
Human CRP was first detected in 1930 and was named for its ability to bind the C polysaccharide of Streptococcus pneumoniae.20 C-reactive protein is mainly synthesized by the liver as part of an acute-phase response after hepatocyte stimulation with proinflammatory cytokines such as interleukin (IL)-6, IL-1, and transforming growth factorβ.21 The function of CRP is not fully understood, but this protein has antiinflammatory properties.2223 Alone or together with procalcitonin, CRP has been a valuable marker for sepsis in critically ill people.2425 In addition, CRP has predictive value in people with atherosclerotic cardiovascular disease.26
In dogs, several studies have shown significant elevations of CRP in experimentally induced inflammation.27–29 However, there are few published clinical studies of CRP concentrations in dogs. In one such study, increased levels of CRP were found in bitches with pyometra.30 In another report, CRP elevations occurred with various disorders and surgical trauma.31 To the authors’ knowledge, the study reported here is the first evaluation of the clinical use of CRP in predicting the presence of pyometra.
The results reported here showed that an elevation in the percentage of bands, in combination with high plasma CRP, was very sensitive in predicting whether the fluid-filled uterus was a pyometra, but the specificity of the combination was low enough (75%) to allow some false-positive results. In contrast, the combination of elevated bands and ALP had a lower sensitivity but a slightly higher specificity.
Using the graph created in the Figure, the line corresponding to the 95% probability predicted the presence of pyometra with high specificity (90%) but also had the increased risk of under-diagnosing pyometra (sensitivity, 77%). In contrast, using the line corresponding to 80% probability had a higher sensitivity (93%) but also had a higher risk of mistaking CEH for pyometra (specificity, 80%). For the clinician trying to decide whether to perform emergency surgery or to wait until a more ideal time, the sensitivity of the test is probably of greater importance than the specificity. It is reasonable to assume that emergency surgery on a dog that is not critically ill (e.g., a dog with CEH) would be less hazardous than the risk associated with a delay in surgery for a dog with impending rupture of the uterus from pyometra. Therefore, it seems logical to use the parameters with the highest sensitivity in predicting the diagnosis. Clinically this requires that a reliable, rapid, and reasonably priced CRP assay be available.
In contrast to WBC counts, CRP assays are not readily available at most small animal practices. The most popular commercially available canine assay today is a relatively expensive and time-consuming ELISA.a In people, automated CRP assays (nephelometric or turbidimetric) are available and are commonly performed 24 hours a day in human intensive care units at an approximate cost of $9.70 (US).24 Structural differences in canine CRP as compared to human CRP have previously precluded the use of human tests in dogs.32 A commercially available, human turbidimetric CRP assay was recently shown to reliably measure canine serum CRP levels, however.33
In addition to the poor availability of CRP to veterinarians, the study reported here revealed a high individual variability in CRP, which made definitive conclusions from a single test challenging.24 Further work is needed to determine the benefits of CRP as a diagnostic aid in veterinary medicine. With increased use of CRP assays in small animals, the development of a readily available, inexpensive automated assay may become a possibility.
In addition to diagnostic laboratory tests, the signalment, history, and physical examination findings are important when creating a list of differential diagnoses. In this study, the age and breed of dog were not helpful in differentiating cases of pyometra from CEH. The average age of the dogs with CEH was slightly younger (6.6 years) than the dogs with pyometra (8.4 years), but the respective ranges were wide and the differences were not statistically significant.
Of the most commonly reported clinical signs in this study, the occurrence of polyuria and/or polydipsia, lethargy, and vomiting and/or inappetence were significantly different between the two groups of diseased dogs. In addition, a dog with only one of these clinical signs was more likely to have CEH, whereas a dog showing three or more signs was more likely to have a pyometra. In this study, physical examination findings had only a limited ability to differentiate pyometra from CEH. General attitude was the only discriminative finding, in that a dog with a bright and alert attitude upon presentation was significantly more likely to have CEH than a pyometra.
In addition to CEH and/or mucometra, pregnancy is another differential diagnosis to pyometra. Similar to pyometra, pregnancy is associated with an enlarged uterus and elevated serum acute-phase proteins.3435 Early pregnancy (2 to 3 weeks) has been associated with elevated levels of haptoglobin, whereas other acute-phase proteins (e.g., ceruloplasmin, glycoprotein, and alpha [2] globulin) have become elevated after 4 weeks of gestation.34 In addition, CRP has been elevated in bitches during midgestation.35 The most dependable diagnostic tool for pregnancy in dogs is ultrasonographic examination of the uterus, which can reliably demonstrate pregnancy 24 to 28 days after breeding.36 In the study reported here, an ultrasound was performed if any suspicion of pregnancy was present. In no case was a pregnancy clinically diagnosed as pyometra; however, in many cases of CEH (n=4) or mucometra (n=4), the diagnosis was often obtained only after histopathological examination of the uterus. Despite the possibility of mistaking elevated acute-phase proteins in pregnancy for pyometra, it would seem that differentiation of these two diagnoses is less of a challenge compared to differentiating pyometra from CEH and/or mucometra, especially when ultrasonography is available.
Conclusion
The percentage of band neutrophils in the WBC count, in combination with CRP concentration, had the highest sensitivity in predicting the presence of pyometra in this study. The other beneficial laboratory parameter for differentiating pyometra from CEH was ALP, which in combination with the percent bands had a slightly increased specificity (at the cost of decreased sensitivity). In addition, the presenting clinical signs and the general attitude of the dog were helpful in the differentiation process. Based on results of this study, guidelines are given for predicting the presence of pyometra versus CEH, and they may have greater relevance in the future with the development of an inexpensive and readily available CRP assay.
Canine CRP ELISA kit; TriDelta Diagnostics, Inc., Plains, NJ 07950
Provided by Dr. W.A. Buurman, University of Maastricht, Maastricht, Netherlands
Microsoft Excel (1997); Microsoft Corporation, Redmond, WA 98052
Proc Logistic (1998); SAS Institute, Inc., Cary, NC 27513
Acknowledgments
The authors express deepest gratitude to Dr. W.A. Buurman, University of Maastricht, Netherlands, for providing reagents for the TNFαassay. The authors also thank Professor Boon Chew for providing laboratory facilities and Bridget Mathiesen for skillful technical laboratory assistance.
Citation: Journal of the American Animal Hospital Association 40, 5; 10.5326/0400391
Contour plot of estimated 95% and 80% probability of the presence of pyometra, based on plasma C-reactive protein (CRP) concentration and the percentage of band neutrophils present in the white blood cell (WBC) count. If values for CRP and bands bisect to the right of the graph, there is a 95% or 80% probability, respectively, that the diagnosis is pyometra in contrast to cystic endometrial hyperplasia (CEH). A fictional case example with 10% bands and CRP of 100 mg/L is included in the graph. In this case, the band and CRP values bisect each other above the dotted line but below the solid line, indicating a probability of pyometra >80% and <95%. Numerical values to the far left and bottom of the graft indicate threshold values for bands and CRP, respectively, above which there is >80% or >95% probability of pyometra.
