VetScan UA Urine Analyzer Clinical Performance Larry Lem, PhD & Andrew Rosenfeld, DVM, ABVP

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1 VetScan UA Urine Analyzer Clinical Performance Larry Lem, PhD & Andrew Rosenfeld, DVM, ABVP Abstract Background: The VetScan UA urine strip reader is an analyzer designed to provide semi-quantitative and qualitative results of common urinalysis chemistries, including microalbumin (MA) and urine protein creatinine (UPC) ratios at the point of care. Objective: This study assessed the precision and accuracy of the VetScan UA semi-quantitative results against a quantitative reference laboratory analyzer. Methods: The precision study evaluated duplicate runs using a total of 126 feline and canine urine samples. Each sample was run twice on two VetScan UA analyzers using VetScan UA14 strips. Clinical Correlation of Urine Chemistries was completed on 108 of the urine samples (40 feline, 68 canine). Samples were run on a VetScan UA analyzer using VetScan UA14 strips and a Roche Automated strip analyzer using Roche Multistix urine reagent strips. Microalbumin correlation was evaluated by comparing the same 108 samples (40 feline, 68 canine) against the Beckman Coulter 2700 Chemistry analyzer, which performs a turbidimetric immunoassay for albumin. The UPC results of the same samples were evaluated against the Beckman Coulter 2700 Chemistry system. Results 1. Precision: The VetScan UA analyzer demonstrated excellent reproducibility for all 13 of the parameters examined. All results were in agreement with the replicate result for all samples tested 99% of the time or more, when defined as identical results or +/- one color block. 2. Chemistry Correlation: Direct comparisons of the VetScan UA results against an automated Roche strip reader for KET, BIL, GLU, PRO, ph, and BLD demonstrated excellent agreement for both canine and feline samples (total n=108). Agreement with the reference method for these results was: 3. Proteinuria Correlation: As proteinuria is a critical indicator of both feline and canine systemic disease, the VetScan microalbumin (MA) and urine protein creatinine ratio (UPC) were evaluated against reference methods. a. Microalbumin (MA) Evaluation: The VetScan MA parameter displayed outstanding agreement with a reference laboratory Beckman Coulter 2700 turbid metric MA assay: b. Urine Protein Creatinine Ratio (UPC) Evaluation: The UPC on the VetScan UA showed excellent accuracy compared to a Beckman Coulter 2700 UPC as follows: Conclusion: All performance indicators examined suggest that the VetScan UA provides excellent precision and accuracy for point-of-care urinalysis chemistry testing. Abaxis VetScan products are intended for veterinary use only. Abaxis and VetScan are registered trademarks of Abaxis, Inc. Abaxis 2018.

2 VetScan UA Urine Analyzer Clinical Performance Larry Lem, PhD & Andrew Rosenfeld, DVM, ABVP Introduction A urinalysis is an important component of a complete clinical diagnostic workup that includes: urine chemistries, gross observations of the urine color and clarity, measurement of specific gravity and an examination of the urinary sediment. The urinary chemistries are important indicators for renal disease, diabetes mellitus, hyperadrenocorticism, hypoadrenocorticism, urinary tract infections and neoplasia. In most veterinary clinic settings, urine chemistries are tested using an inexpensive, disposable urine reagent strip. These urine strips contain a set of absorbent pads, each embedded with chemicals that react with urine components to provide colorimetric changes designed to either be interpreted by the user against a color chart or in some cases measured by an instrument. Analytes measured typically include: bilirubin (BIL), glucose (GLU), protein (PRO), specific gravity (SG), ph, and blood/hemoglobin (BLD). The analyte concentrations are derived from color changes on the pads that are induced by chemical reactions with the analytes. For traditional urine test strips, analyte concentrations are determined through a color comparison of the exposed strip to a reference color chart. The advantages of such a test method are that the testing is simple and no instrumentation is required. However, this traditional testing process contains a number of sources for potential variability in the results, including: subjective interpretation of the color shade, variability of lighting in the room, the amount of urine left on the strip during color development, potential contamination from other pads during color development, the presence of interfering substances (such as blood/hemoglobin), and the amount of time the strip is exposed to urine prior to the color chart interpretation. Each pad has a specific reaction time after which the pad should be read. On a single strip, 3-4 different optimal reaction times may be present. Reading the strip at an inappropriate time, or a delay in reading the strip, can lead to under or overdevelopment of the chemical color change on the pads and thus lead to inaccurate results. Due to the variability inherent to the traditional urine strips testing process, all urine strips report semi-quantitative results, defined as reporting results in a series of ranges or bins to represent the entire range of concentrations. Each bin represents an analyte concentration range (for example from 45 to 88 units, see Figure 1) and is named with both a symbol and numerical concentration value, such as +2 and 70 mg/dl as in the example in Figure 1. The exact color gradation on a given test pad is used to calculate the analyte concentration via an algorithm determined by the test strip manufacturer. Any test run whose calculated concentration falls within the range of a bin will have a reported result corresponding to that bin s symbol and numerical concentration value. Any two calculated results falling within a bin are reported to the user as the same value, despite the fact that the actual colors of the two pads are slightly different, as shown in the example of Pair A in Figure. Analyte Concentration (mg/dl) Figure 1. Semi-Quantitative Results. The graph illustrates how semi-quantitative results are generated for five bins. Pair A shows two samples calculated to have a concentration of 50 and 86, which will both be reported in the same bin (70 ml/dl or 2+) to the user. Pair B illustrates how two close test results can sometimes fall into separate bins. The precision of semi-quantitative results are typically evaluated by having acceptable limits of either one bin over or one bin below the actual bin. This concept is illustrated by Pair B in Figure 1, where the first calculated result is 86 units, which falls within the bin of 70, and thus reported to the user as 70 or 2+. If a replicate test is run on the same sample with a new strip, the second result may be 91 and thus reported as 125 or 3+. The actual colors of the pads in the two runs and the actual concentrations of analytes are very close to each other despite the names of the two bins, 70 and 125, appearing to be far apart. Abaxis has introduced the VetScan UA analyzer system that removes much of the variability in urine chemistry strip testing through an automated scanning of test strips at precise times to prevent under or overdevelopment and subjectivity of evaluating the color change of the 1 of 5

3 pads. The VetScan UA analyzer also provides the ability to semi-quantitatively measure microalbumin (MA) levels and calculate a urine protein creatinine ratio (UPC). Both MA and UPC are indicators of proteinuria, which is an important prognostic indicator and monitoring tool for renal disease in both dogs and cats (1) (2). MA levels above 2.5 mg/dl (25 mg/l) suggest that glomerular protein leakage is clinically significant. While a single high MA result may occur due to transient physiological changes, such as an acute inflammatory process, repeated high MA levels over a period of weeks may be suggestive of early renal disease or a chronic inflammatory condition requiring further diagnostic investigation (3). The UPC provides a good method to quantify renal proteinuria and is a key indicator for staging a dog or cat for chronic kidney disease (3). UPC values above 0.4 (feline) or 0.5 (canine) indicate leakage of protein from the kidneys at levels which may indicate renal dysfunction, and require monitoring for potential progression of renal disease (3). A UPC over 1.0 requires diagnostic and potentially therapeutic intervention, and a UPC over 2.0 is usually associated with overt glomerular proteinuria (3). This study examines the accuracy and precision of the VetScan UA analyzer against reference laboratory analyzers for traditional urine chemistries on feline and canine urine samples. As the VetScan UA tests for both MA and UPC on its UA14 urine test strip, this study also examined how the VetScan UA UPC and MA compared to a reference laboratory analyzer. Materials and Methods Calibration: Analyzers were checked for correct calibration using VetScan UA controls and VetScan UA Check strips daily prior to data collection. Precision: The precision study evaluated duplicate sample runs using a total of 126 urine samples that included both canine and feline samples. Samples were run twice on each of two VetScan UA analyzers using VetScan UA14 strips. Replicate runs were run immediately after the first run on each sample. Sample runs were considered to be repeatable if the results of the two duplicate runs were within one color block (either up or down, but not both) of the replicate sample results. The number of identical results and results +/- one block between the two replicate runs on both analyzers were tallied and calculated as a percentage of the total sample runs. Clinical Correlation of Urine Chemistries: 108 urine samples (40 feline, 68 canine) were run on a VetScan UA analyzer using VetScan UA14 strips and a Roche Automated strip analyzer using Roche Multistix urine reagent strips. Samples were run on either the Roche analyzer or VetScan UA analyzer within one hour of running on the other analyzer. The VetScan UA urine chemistry results were compared to those results for the following parameters: KET, BIL, GLU, PRO, ph, and BLD. Data for LEU, NIT, URO, ASC and Ca were collected for information purposes only and not evaluated in this study since the parameters are not relevant in veterinary medicine. The SG measurement was also not evaluated, as SG evaluation on a refractometer is recommended. The results were rated in similar bins to facilitate comparison. The VetScan UA results were then compared as to whether they were in agreement ( IA for In Agreement, defined as identical or within one bin of the Roche results). Results greater than one bin away were classified as out of range (OOR). The numbers of samples in agreement were described as a percentage of the total number of samples. Microalbumin (MA) Evaluation: To evaluate the accuracy of the microalbumin (MA) results of the VetScan UA analyzer, the results from 108 urine samples (40 feline, 68 canine) all performed on the same analyzer were compared to results from a reference laboratory Beckman Coulter 2700 Chemistry analyzer, which performs a turbidimetric immunoassay for albumin. Since the VetScan UA analyzer uses a threshold of 2.5 mg/dl (25 g/l) to indicate a positive MA result, the quantitative Beckman MA results were rated as to whether the results were greater than 2.5 mg/dl (25 g/l) and compared with the VetScan UA results. Information on the health of each patient was not available for this study. Urine Protein Creatinine Ratio (UPC) Evaluation: The UPC results of the VetScan UA analyzer were evaluated against a reference laboratory Beckman Coulter 2700 Chemistry analyzer. 108 samples (40 cat samples, 68 dog samples) were run between the VetScan UA and Beckman Coulter systems. The UPC results on the VetScan UA analyzer were categorized into 3 bins for cats and dogs, respectively: between 0.0 and 0.4/0.5, between 0.4/0.5 and 2.0, and >2.0. The 0.4/0.5 UPC threshold was selected according to the International Renal Interest Society Chronic Kidney Disease staging guidelines (3). These were matched against the results of the Beckman Coulter analyzer and then categorized as false positive or false negative to generate the sensitivity and specificity ratings. Statements relating to UPC thresholds of 0.5 are intended to include feline samples for which the threshold is 0.4. Results Precision: The VetScan UA analyzer demonstrated excellent reproducibility for all 13 of the parameters examined. All results were in agreement with the replicate result for all samples tested 99% of the time or more, when defined as identical results or +/- one color block (Table 1). This held true across all 13 parameters. Identical results were seen between the replicate runs, ranging from 75% to 100% of the samples. For example, 100.0% of the replicate PRO results were in agreement, with 91.3% of results being identical between the pairs and 8.7% being +/- one color block from the other replicate. Table 1. VetScan UA Precision on VetScan UA14 Strips Urine Samples 2 of 5

4 Clinical Correlation General Urine Chemistries: The VetScan UA results compared very well to the Roche automated strip analyzer for the urine chemistries. For the most critical analytes shared between the VetScan UA and the Roche analyzers(bil, GLU, PRO, ph and BLD) the percent of samples in agreement (IA) were 100%, 99%, 93%, 90% and 92%, respectively (Table 2). Only 0.9% of samples on the VetScan UA analyzer were out of range (OOR) compared to GLU results from the Roche. Out of range results ranged from 7.4%, 10.2% and 8.3% for PRO, ph and BLD, respectively. The VetScan UA demonstrated complete agreement with the Roche analyzer for all BIL results tested. results being within the healthy range, when the reference result was in a higher bin. Performance for UPC on feline and canine samples were similar in the percentage of false positives and negatives (data not shown). Table 2. Agreement between VetScan UA Chemistries and a Roche Automated Strip Reader (n=109 samples, run on two analyzers, total of 216 comparisons) The VetScan UA demonstrated excellent sensitivity (87%) as it detected 65 of 75 samples as non-proteinuric, as defined by a UPC value below 0.4 (feline) or 0.5 (canine). The VetScan UA analyzer also provided results in agreement in 22 of 28 samples in the borderline proteinuric category. The VetScan UA results were in 100% agreement for the 5 samples that showed a UPC above 2.0 on the reference analyzer. Microalbumin (MA): The VetScan UA MA results were compared to the reference analyzer for 108 samples matched the reference analyzer extremely well. 85% of the samples were in agreement between the two analyzers as either being above or below the 2.5 mg/dl (25 mg/l) MA threshold which indicates a positive test for microalbuminuria. Of the 51 MA positive samples per the reference analyzer (MA>2.5 mg/dl), MA was detected in 49 samples by the VetScan UA analyzer (sensitivity 96.23%), showing that the VetScan UA accurately indicated protein leakage (Table 3). The VetScan UA produced 12 false positives in 108 samples for a specificity of 82.61%. The VetScan UA only produced 2 false negatives in this sample set. Performances for MA on feline and canine samples were similar in the percentage of false positives and negatives (data not shown). Table 3. Evaluation of Microalbumin (MA) Results vs Reference Analyzer. FP, FN are False Positives and False Negative, respectively. Table 4. Evaluation of UPC Results vs Reference Analyzer. Clinical Use of MA and UPC The clinical utility of the microalbumin (MA) and urine protein creatinine ratio (UPC) were further examined in conjunction with each other. Microalbuminuria indicates the early leakage of micro-amounts of protein into the urine, usually preceding a rise in UPC value. Healthy animals should not have any proteinuria (MA<2.5, UPC<0.5). In early renal disease, microalbumin leakage will begin to increase before UPC reaches significant levels (MA+, UPC<0.5). As disease develops, the UPC levels will begin to rise above 0.5 and the MA readout loses its diagnostic value as renal disease has become overt and can be detected by UPC. To examine how the Vetscan UA UPC measurements performed relative to a reference analyzer regarding the correlation of UPC and MA, they were plotted against the reference analyzer MA for the same sample. As expected, samples with MA of less than 2.5 mg/dl (25 mg/l) corresponded with a UPC value lower than 0.5 (Figure 2). Four samples on the VetScan UA resulted in a UPC between 0.5 and 2.0 with MA < 0.5. Three of those samples resulted in UPC levels on the reference analyzer of 0.1 and one had a UPC of 0.2 (Table 5). Thus, the UPC reference analyzer results for all four samples were borderline to an adjacent bin and were only one bin away from the results seen on the VetScan UA. Table 5. Reference Analyzer Results (Beckman Coulter 2700 Chemistry Analyzer, BC) for Four MA-UPC Level 3 ( ) Results Urine Protein Creatinine Ratio (UPC): The VetScan UA UPC results showed very good agreement with the results of the reference analyzer. 92% of VetScan UA UPC results were in exact agreement or within one bin of the reference analyzer result. Previous studies and IRIS guidelines have determined that healthy cats and dogs have UPC ratios below 0.4 or 0.5, respectively (3). The number of results from the two analyzers falling above and below that critical UPC limit (0.4 or 0.5) (3) was examined (see Table 4). (Henceforth, references to the 0.5 UPC for dogs will assume the feline UPC threshold of 0.4 as well.) False Positives (FP) are defined as Vetscan UA results reading above 0.5 when the reference result was below 0.5 and False Negatives (FN) are defined as VetScan UA 3 of 5 As MA levels increased, the UPC typically increased, as expected. Eighteen samples displayed positive MA results with UPC<0.5 on the reference analyzers, but only at the lower end of MA concentrations. The presence of such MA positive, UPC <0.5 samples is consistent with previous studies (4). All of these observations are consistent with the expectations of the development of renal disease.

5 Examining the data from the reference analyzers in a similar manner produced matching results with the VetScan UA data, indicating good clinical agreement between high end reference analyzers and the VetScan UA when both UPC and MA are considered together (Figure 3). An alternative graphical comparison of the MA and UPC results was examined to determine if the VetScan UA produced equivalent diagnostic results when both parameters were considered simultaneously. Figure 4 shows that the VetScan UA produced results that closely matched the reference analyzer. The number of samples above the clinically significant UPC limit of 0.5 was plotted when the samples had MA>2.5 mg/dl (MA+) or MA<2.5 mg/dl (MA- ). The VetScan UA closely matched the number of samples on the reference analyzers for MA+, UPC , in addition to MA+, UPC>2.0. This data indicates that the VetScan UA agrees well with reference analyzers when the MA and UPC parameters are evaluated together for clinical diagnosis of proteinuria. The VetScan UA results labeled more samples as MA+, UPC<0.5 than the reference analyzers did (Figure 4B), but a closer examination of these showed that the VetScan UA differed from the Beckman Coulter results by only one bin in all but two of these cases and thus were considered within agreement levels. In addition, any MA+ result should be repeated in 2-3 weeks prior to making a diagnosis. Conclusions Urine chemistry testing is an important point-of-care diagnostic tool for screening and monitoring renal disease in veterinary patients. The VetScan UA was evaluated for precision and accuracy in traditional urine chemistries as well as two newly available point-of-care parameters, microalbumin and the urine protein creatinine ratio. The VetScan UA showed excellent precision on results from the UA14 strips for the most commonly tested urine chemistry analytes, with matching results in 99% of replicates. The Vetscan UA also demonstrated excellent accuracy compared to a reference automated urine strip reader for all the analytes tested, including MA and UPC. Despite the fact that the reference analyzers provide quantitative results, while the VetScan UA produces semi-quantitative results, the results correlated well. For the two novel parameters on a urine test strip, both microalbumin and urine protein creatinine ratios showed very good agreement with the reference analyzer results. Data indicates that the VetScan UA generated very few false negative MA results, and thus is an effective screening tool for low level proteinuria and early stage renal disease. When MA and UPC on the VetScan UA are used in combination, they provide a powerful screening tool for renal disease that correlates well with results from reference laboratory analyzers. References: 1. Survival of cats with naturally occurring chronic renal failure is related to severity of proteinuria. Syme HM, Markwell PJ, Pfeiffer D, Elliott J. 3, May-Jun 2006, J Vet Intern Med., Vol. 20, pp Evaluation of the association between initial proteinuria and morbidity rate or death in dogs with naturally occurring chronic renal failure. Jacob F, Polzin DJ, Osborne CA, Neaton JD, Kirk CA, Allen TA, Swanson LL. 3, Feb 1, 2005, J Am Vet Med Assoc., Vol. 226, pp IRIS CKD Staging Guidelines. [Online] Nov 17, [Cited: Nov 17, 2017.] 4. Comparison of a semiquantitative point-of-care assay for the detection of canine microalbuminuria with routine semiquantitative methods for proteinuria. Garner, BC, Wiedmeyer, CE. 3, s.l. : Vet Clin Path, 2007, Vol. 36, pp Figure 2. Combination of UPC and MA in Clinical Diagnosis for the VetScan UA. UPC results on the UA were categorized into 4 levels (Level 1, 2, 3 and 4 corresponding to <0.2, , , >2.0, respectively, and graphed versus MA on the reference analyzer (Beckman Coulter 2700). Dashed lines indicate clinical thresholds of 0.4 (feline)/0.5 (canine) UPC and 2.5 mg/dl MA for felines and canines. 4 of 5

6 Figure 3. Combination of UPC and MA in Clinical Diagnosis for the reference laboratory analyzer (Beckman Coulter 2700). Dashed lines indicate the clinical thresholds of 0.4 (feline)/0.5 (canine) UPC and 2.5 mg/dl MA for felines and canines.. (A) No. of Samples < >2.0 UPC UA MA- Ref MA- (B) No. of Samples < >2.0 UPC UA MA+ Ref MA+ Figure 4. The Diagnostic Combination of UPC and MA on the VetScan UA. UPC and MA closely matches those on reference lab chemistry analyzer. (A) Plot of number of MA negative samples versus UPC results on the VetScan UA and reference analyzer (Beckman Counter 2700) shows similar numbers of samples in each category for this set of samples. (B) Plot of number of MA positive samples versus UPC results on the VetScan UA and reference analyzers (Beckman Counter 2700 and Roche analyzers) shows similar numbers of samples in each category for this set of samples. 5 of 5 Abaxis VetScan products are intended for veterinary use only. Abaxis and VetScan are registered trademarks of Abaxis, Inc. Abaxis Rev. B