Effect of feline characteristics on plasma N-terminal-prohormone B-type natriuretic peptide concentration and comparison of a point-of-care test and an ELISA test

2.1 Recruitment

The study was approved by the Uppsala Animal Experiment Ethics Board. Cats were recruited prospectively to participate in the study through information for cat owners provided on webpages, at seminars or at the recruiting clinic. Client-owned cats were examined at Evidensia Animal Clinic in Västerås between September 2014 and June 2017. Clinically healthy cats and cats with murmurs or previously diagnosed HCM were examined for possible inclusion in the study. Informed written consent was obtained from the owner of each cat.

2.2 Inclusion criteria

Healthy cats of nonpurebred Domestic Shorthair (DSH), purebred Birman and Norwegian Forest (NF), and HCM cats of any breed were allowed into the study. Cats with preclinical or clinical HCM, stabilized as a consequence of CHF treatment, were allowed into the study. Cats aged between 1 and 14 years were eligible for the study. All healthy cats must have had normal echocardiogram. Diagnosis of HCM was based on characteristic findings on an echocardiogram as outlined below.^21-23

2.3 Exclusion criteria

Cats with increased mean systolic blood pressure (SBP) >160 mmHg, increased serum concentrations of thyroxine (TT4), creatinine, or fructosamine, increased alanine aminotransferase (ALT) activity, or some combination of these were excluded from the study. Cats with decompensated CHF, thromboembolism, congenital cardiac disease, other acquired cardiovascular disorders, equivocal findings concerning the presence of hypertrophy of the LV, severe dental disease or clinically relevant organ-related or systemic diseases were not included in the study. All cats receiving medical treatment other than those in need of standard CHF treatment, standard antithrombotic treatment, medroxyprogesterone acetate, or deslorelin acetate were excluded from the study.

2.4 Clinical examination and blood pressure measurement

All examinations were performed according to a standardized protocol in a quiet examination room by a single experienced veterinarian (S.H.). The healthy control cats were examined between 0900 and 1300 hours. The cats were allowed to adapt to the environment for 10-15 minutes together with their owners before SBP measurement.²⁴ An appropriate cuff was applied to the base of the tail and the cats then were allowed to rest in their carrier before SBP was indirectly measured using an automated high-definition oscillometric (HDO) device (VET Memodiagnostic HDO monitor, S+B medVET, Babenhausen, Germany).²⁵ Once consistent consecutive readings were obtained, 6 recordings were performed. Mean values for SBP were determined for each cat, after discarding recordings deviating more than 20%.²⁴

After the SBP measurement, all cats underwent general physical examination. The BCS was recorded on a 9-point scale.²⁶

2.5 Echocardiography

Transthoracic 2-dimensional (2D), M-mode, color flow, and spectral Doppler echocardiographic evaluations were performed by an experienced echocardiographer, trained by a board-certified veterinary cardiologist, in unsedated cats placed first in right and then in left lateral recumbency using an ultrasound system (IE33, Philips Ultrasound, Bothell, Washington) equipped with a 4-12 MHz phased-array probe. Continuous electrocardiography monitoring was performed during echocardiographic examination. Images and loops from standardized imaging planes²¹ were stored digitally. The left atrial-to-aortic root diameter ratio (LA/Ao) was measured in 2D from the right 2D short-axis view.²² Diastolic and systolic LV dimensions (interventricular septum diastole [IVSd], LV internal diameter in end diastole [LVIDd], LV free wall diameter in end diastole [LVFWd], LV internal diameter in systole [LVIDs], and fractional shortening [FS]) were measured from M-mode and 2D using right parasternal short- and long-axis images.^{21, 23} Mitral, tricuspid, aortic, and pulmonic valves were interrogated using spectral and color-flow Doppler according to published recommendations.^{21, 23} Two-dimensional images of the LV outflow tract were used to identify the presence of systolic anterior motion (SAM) of the mitral valve.

The diagnosis HCM was made when a subjective impression of hypertrophy (regional or global) was supported by increased M-mode and 2D diastolic LV wall dimensions of the interventricular septum (IVSd), left ventricular free wall (LVFWd), or both defined as outlined below. Left atrial size was assessed using the LA/Ao as previously described.²² Expected BW-dependent values for IVSd, LVIDd, and LVFWd were calculated according to previously generated formulas for cats.²⁷ Percentage deviation from these expected values was calculated according to the formula: X_inc(%) = ([observed value-expected value]/expected value) × 100, where X represents the variable in question. These calculated percentage deviations from expected BW-dependent values,²⁷ and the LA/Ao ratio were used to classify the cats into 3 different groups: healthy controls, HCM without LAE, and HCM with LAE. Cats with subjectively normal LV morphology, LA/Ao <1.5,^{27, 28} and <30% increase in BW-based predicted values for IVSd and LVFWd²⁷ were classified as healthy cats. Cats with subjective impression of LV hypertrophy and ≥30% increase in BW-based predicted values for IVSd or LVFWd were classified as having HCM without LAE if LA/Ao <1.5, and with LAE if LA/Ao ≥1.5. Cats that could not be classified according to the criteria specified above were excluded from the study.

2.6 Blood collection and analysis

Blood was collected in ethylenediaminetetraacetic acid (EDTA) and serum tubes by venipuncture of the cephalic vein in unsedated minimally restrained cats. Hematology and serum biochemistry profiles, including ALT activity, creatinine, glucose, and total protein concentrations, were performed at Evidensia Animal Clinic in Västerås immediately after sampling using the ProCyte (IDEXX ProCyte Dx, IDEXX Laboratories, Inc) and Catalyst (Catalyst Dx Chemistry Analyzer, IDEXX Laboratories, Inc) systems. Total thyroxine and fructosamine concentrations were analyzed within a few days after each sampling at the Clinical Pathology Laboratory of the University Animal Hospital, Swedish University of Agricultural Sciences using the Immulite (IMMULITE 2000, Siemens Healthcare GmbH, Erlangen, Germany) and Abbott Architect (Abbott Architect c4000, Abbott Park, IL, USA) systems.

For later analysis of NT-proBNP, EDTA blood was centrifuged and plasma frozen at −80°C within 30 minutes of collection for storage. All samples were transported frozen (−80°C) to a commercial laboratory (Vet Med Labor GmbH, Division of IDEXX Laboratories, Ludwigsburg, Germany) for batch analysis in duplicate using a validated second-generation ELISA for cats (CardioPet NT-proBNP assay, IDEXX Laboratories, Inc).²⁹ The reported assay range for NT-proBNP concentration at the commercial laboratory was 24-1500 pmol/L. For statistical analyses, concentrations of NT-proBNP less than the lowest reported concentration were assigned a result of 24 pmol/L. Concentrations of NT-proBNP >1500 pmol/L were assigned a result of 1500 pmol/L.

All samples also were analyzed using the POCT feline NT-proBNP (SNAP Feline proBNP, IDEXX Laboratories Inc) according to the instructions of the manufacturer. The samples were randomized and the examiner (S.H.) performing the evaluation of the POCT was blinded to the identity of the cats. Plasma was thawed and kept at room temperature for <30 minutes before analysis. Normal results were recorded when the density of the sample spot appeared lighter than the reference spot, according to the manufacturer's instructions (IDEXX Reference Laboratories Inc). Abnormal results were recorded when the density of the sample spot appeared equal to or darker than the reference spot, which, according to the manufacturer, occurs when concentrations are >100 pmol/L (IDEXX Reference Laboratories Inc). Evaluation of the POCT was performed on the same occasion by visual inspection and using an automated evaluation (SNAP Pro Analyzer, IDEXX Laboratories Inc). The examiner performed the visual inspection before checking and documenting the results from the automated evaluation. Visual inspection of the POCT results was classified into 3 groups (lighter, equal, and darker), based on subjective evaluation of the sample spot color. After all samples had been analyzed, concentrations of NT-proBNP measured by ELISA were revealed.

2.7 Statistical analysis

Statistical analyses were performed using a commercially available software program (JMP, version 12.2.0, SAS Institute Inc, Cary, North Carolina). Group data are presented as medians and interquartile range (IQR). A value of P < .05 was considered significant, unless otherwise indicated.

Univariable analyses were performed separately in 3 groups of cats: all cats, healthy cats and HCM cats. Fisher's exact test was used for comparing proportions. The nonparametric Kruskal-Wallis test was used to evaluate the effects of nominal and ordinal variables (sex, neutered or intact, BCS, presence or absence of SAM) on NT-proBNP concentrations. Furthermore, the Kruskal-Wallis test was used to evaluate the overall association between the POCT results (intensity of color in the POCT) and the plasma NT-proBNP concentrations in all cats, and for evaluating the effect of breed in the healthy cat group. The Kruskal-Wallis test was used followed by Bonferroni correction for post hoc comparison between groups. These statistical analyses were repeated in the HCM cats after excluding 6 cats treated with cardiac medications (furosemide, enalapril, clopidogrel).

Spearman's ρ was used to evaluate potential associations between plasma NT-proBNP concentration and continuous variables (age, SBP, heart rate auscultation [HR] obtained from clinical examination, echocardiographic measurements, and storage time of the plasma samples) for any of the cat groups with a high rate of nondetectable NT-proBNP plasma concentrations.

After logarithmic transformation of the concentration of NT-proBNP, linear regression was used to evaluate potential associations between plasma NT-proBNP concentration and continuous variables (age, BW, SBP, echocardiographic measurements, HR obtained from clinical examination, and storage time of the plasma samples) in any of the cat groups with a high rate of detectable NT-proBNP plasma concentrations. Variables with P < .2 in the univariable regression analysis were included in a multiple regression analysis as were BCS and sex. Multivariable analyses were performed in a backward stepwise manner, starting with all variables included in the model and then removing the variable with the highest P value until all of the remaining variables had a P value <.05. All variables were assessed as main effects; no interaction terms were considered in the model. The distribution of the residuals was assessed for normality by inspection of normal quantile plots. The adjusted R ² is defined as the percentage of the total sum of squares that can be explained by the regression and it also considers the degrees of freedom for variables added.

The optimal cutoff (combination of highest SE and SP) in NT-proBNP concentration for identifying an abnormal POCT was investigated using receiver operator characteristic (ROC) curves.

3.1 Study population

Of 118 examined apparently healthy cats, 18 cats were excluded. Five of these cats had kidney disease, 5 had mild heart disease (4 cats had cardiac disease other than HCM and 1 cat had equivocal findings concerning presence of hypertrophy of the LV), 2 had severe dental disease, 2 had congenital defects (diaphragmatic hernia and peritoneal-pericardial diaphragmatic hernia), 2 had ALT activity above the reference range, and 1 had hyperthyroidism. Of 48 examined cats with murmurs or previously diagnosed HCM, 9 were excluded: 2 of these cats were normal, but did not meet the breed criteria for the healthy controls, 2 had congenital defects (ventricular septal defect and bicuspid Ao), 1 had equivocal findings concerning presence of hypertrophy of the LV, 1 was in decompensated CHF, 1 had kidney disease, 1 had hyperthyroidism, and 1 cat was treated with 4 times the normal dose of clopidogrel.

A total of 139 cats (69 males and 70 females) were included in the study: 100 healthy controls, 32 cats with HCM without LAE and 7 HCM cats with LAE. Three of the cats with HCM had experienced CHF and were stabilized by medical treatment. The healthy cats consisted of 35 NF, 33 Birman, and 32 DSH cats. The HCM cats comprised 13 breeds: 18 nonpurebred DSH, 5 Persian, 4 NF, 2 Bengal, 2 Maine Coon, 2 Ragdoll, and 1 each of the following breeds: British Shorthair, Cornish Rex, Exotic, Siberian, and Sphynx. Two healthy male cats were treated with deslorelin, and 1 of the female cats with HCM without LAE was treated with medroxyprogesterone acetate. Three of the cats with HCM without LAE were treated with enalapril, and 3 of the stabilized CHF cats were treated with furosemide and enalapril, of which 1 cat also received clopidogrel. Feline characteristics, concentration of NT-proBNP, and echocardiographic measurements of the 3 cat groups (healthy, HCM without LAE, and HCM with LAE) are presented in Table 1 and Figure 1. Feline characteristics, echocardiographic measurements, concentration of NT-proBNP, and POCT results of the healthy control cats of the 3 different breeds are presented in Table 2.

Blood glucose concentration was mildly increased in 11/139 cats. However, serum fructosamine concentrations were measured and were within normal limits in all cats, as were hematology and other serum biochemistry variables.

3.2 Evaluation of NT-proBNP plasma concentration measured by ELISA

The intra-assay coefficient of variation (CV) for the feline NT-proBNP ELISA was 5.2%-8.1%, and the interassay CVs were 7.2, 3.1, and 4.2%, respectively, for samples with mean concentrations of 57, 229, and 639 pmol/L, respectively.

3.3 All cats

3.4 Healthy cats

The median NT-proBNP concentration in the healthy cats was <24 pmol/L in all 3 breeds with similar IQR across the breeds (Birman, <24-39; DSH, <24-38; NF, <24-29 pmol/L). Three of the healthy controls had concentrations >100 pmol/L (110, 117, and 155 pmol/L, respectively).

3.5 Cats with HCM

Of the 39 cats with HCM, 11 cats had an NT-proBNP concentration <100 pmol/L (range, <24-69 pmol/L).

3.6 Evaluation of POCT NT-proBNP test results

3.6.2 Healthy cats

By visual inspection of the POCT, 98/100 healthy cats were classified as normal and 2/100 as abnormal (ELISA NT-proBNP concentrations in these cats were 110 and 155 pmol/L, respectively; Figures 2 and 3).

By automated evaluation of the POCT, 95/100 healthy cats were evaluated as normal and 5/100 healthy cats as abnormal (corresponding ELISA NT-proBNP concentrations in these cats were 49, 61, 78, 88, and 155 pmol/L, respectively).

4.1 Study Limitations

Blood was collected only once from each cat. Therefore, biological variation in NT-proBNP concentration within an individual cat could not be evaluated. Studies have indicated a high biological variation for NT-proBNP concentrations in cats, dogs, and humans, and this variability may affect the SE and SP.^47-49 The healthy control cats were examined in the morning to decrease the effect of diurnal variation among the cats included in the study. Cats with HCM were not examined at a standardized time of day because of the clinical condition of the cat and for practical reasons.

Plasma samples were stored at −80°C up to 4 years before batched analyses were performed, but storage time was not shown to be associated with the NT-proBNP plasma concentration. Furthermore, previous studies have shown that NT-proBNP concentrations are stable at the freezing temperatures used in our study.^50-52 It thus is unlikely that storage had any major effect on the results.

The POCT becomes abnormal within a transition interval rather than a set cutoff value, which is a limitation. In our study, the transition interval was found to between 69 and 117 pmol/L, which is slightly lower than a previous study in which the transition occurred between 108 and 122 pmol/L⁹ but different from another study in which the transition interval was between 150 and 200 pmol/L.¹¹ However, the SE and the SP for the ELISA and the POCT were similar, therefore the POCT results seem reliable.

In our study, the cats with HCM without LAE generally were mildly affected and few cats had HCM with LAE. Furthermore, a few of the cats included were treated with deslorelin, medroxyprogesterone acetate, or cardiac medications. Three of the HCM cats with LAE were on medical treatment for previous CHF, and plasma concentrations of NT-proBNP in cats treated for CHF may decrease after stabilization of CHF.⁵³

The design of our study, using selected inclusion and exclusion criteria, may have influenced the accuracy of the test and thereby overestimated SE and SP. Our study was intended as an explorative study, and further research in a mixed cat population is warranted.

Although no differences in NT-proBNP concentration were found among the 3 genetically distant breeds in the study, differences among other breeds cannot be excluded.