Comparison of Home Monitoring Methods for Feline Urine pH Measurement Rose E. Raskin, Kelly A. Murray, Julie K. Levy Background — Monitoring of urine pH, often done in the patient’s home, is essential for proper clinical treatment and management of conditions such as urolithiasis. Objective — The purpose of this study was to assess the agreement in pH readings between a standard laboratory method and methods readily available for home monitoring. The influence of refrigerated storage on urine pH was also examined. Methods — Urine samples were obtained by cystocentesis from 40 clinically healthy cats, and pH was measured within 2 hours of collection. Each sample was evaluated using pH paper, urinalysis reagent strip, 2 brands of portable pH meters (Chek-Mite, Corning, Corning, NY, USA; and Checker 1, Hanna Instruments, Woonsocket, RI, USA), and a standard laboratory benchtop pH meter. Urine samples were refrigerated, and a second pH reading was obtained with the laboratory benchtop meter after 24 hours. The degree of agreement was assessed among the different methods, with the laboratory benchtop pH meter as the reference method. Results — The closest agreement was obtained with the Chek-Mite portable pH meter and least agreement with the Checker 1 portable pH meter, which had a constant negative bias of 0.31 units due to expiration of the electrode. As expected, pH paper and reagent strips had poor and intermediate agreement, respectively. The reagent strip method had a negative bias of 0.12 units when compared with the benchtop pH meter and wide disagreement at the low pH end. The reagent strip did not agree strongly with the reference method; only 50% of values were within 0.25 pH units of each other. The difference in pH between 0 hours (6.57 ± 0.54) and 24 hours of refrigeration (6.61 ± 0.53) was not considered clinically significant. Conclusion — Portable pH meters are excellent for monitoring urine pH at home as long as attention is given to electrode maintenance. Urine can be collected at home and kept refrigerated, and pH may be measured reliably within 24 hours using the reference method or a portable pH meter. (Vet Clin Pathol. 2002;31:51-55) Key Words: Cat, instrumentation, method comparison, pH, urinalysis, urine

———◆——— Monitoring of urine pH is essential for proper clinical treatment and management of urolith formation. Several variables affect urine pH such as food intake, diet, age, and breed. After ingestion of a meal, urine pH may increase because of postprandial alkaline tide, which is related to increased gastric acid secretions needed for digestion.1 Protein, minerals, and organic ions in the diet also affect urine pH and are manipulated in special diets used to manage certain uroliths. Feline urolithiasis is an important and difficult disease syndrome to manage medically. Dietary management of stone formation has been an important therapeutic option.2 Careful monitoring of urine pH is used to assess accuracy of treatment but can be problematic with excited cats. In a cat with chronic urolithiasis that was being treated with an acidifying diet, the urine had struvite crystals and alkaline pH readings, as measured by a pH meter.3 The veterinarian determined that the stress of a clinic visit caused hyperventilation and subsequent acute respiratory alkalosis, resulting in alkaline urine. A cat with such a reaction to clinical examination would be an ideal candidate for home measurement of urine pH with a portable pH meter. There are several methods used to measure pH,

including urine reagent strips (dipsticks), pH paper, and pH meters. There are 2 types of pH meters: standard benchtop models and more convenient, less expensive portable models. The use of portable pH meters was evaluated recently in food animals, where they were reliable for measuring the pH of rumen fluid and urine.4 The purpose of this study was to compare various methods for home monitoring of feline urine pH. Because in some cases home monitoring might not be possible and transport of urine to a clinic would be necessary, we also examined whether refrigeration of urine over a 24-hour period had an effect on pH. Materials and Methods Five-milliliter urine samples were obtained by cystocentesis from 40 sedated cats involved in the University of Florida College of Veterinary Medicine feral cat spay/ castration clinic (Operation Catnip). An equal number of females and males were used, ages 6 months to 2 years. Medical history was not known, but all cats appeared clinically healthy. Urine color was recorded at the time of collection. Urine pH was measured within 2 hours of collection,

From the Departments of Physiological Sciences (Raskin, Murray) and Small Animal Clinical Sciences (Levy), College of Veterinary Medicine, University of Florida, Gainesville, Fla. Corresponding author: Rose E. Raskin, DVM, PhD, PO Box 100103, Veterinary Medical Teaching Hospital, University of Florida, Gainesville, FL 32610-0103 ([email protected]).

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Table 1. Comparison of methods used to measure urine pH in 40 cats Method

Mean

SD

SEM

95% Confidence Interval

Median

Minimum

Maximum

pH paper

6.4

0.58

0.09

6.18-6.55

6.00

6.0

8.0

Dipstick

6.5

0.53

0.08

6.28-6.62

6.50

6.0

8.0

Chek-Mite portable meter

6.58

0.494

0.078

6.42-6.74

6.490

5.71

7.71

Checker portable meter

6.27

0.540

0.085

6.09-6.44

6.255

5.49

7.64

Benchtop meter

6.57

0.539

0.085

6.40-6.74

6.555

5.81

8.01

Instruments, Woonsocket, RI, USA), and a standard benchtop pH meter (pH meter 220, Corning). A small amount of urine was placed on pH paper and dipsticks, and the pH was visually estimated to the nearest 1 pH unit (pH paper) or the nearest 0.5 pH unit (dipsticks). The same individual interpreted the color reaction, thereby eliminating interobserver variability.The manufacturers’ instructions indicated an accuracy of ±1.0 pH units in the range of pH 5.0-8.5 for the pH paper and for the dipstick. Two-point calibration was performed initially for the portable and benchtop pH meters using A commercial buffers of pH 4.0 and 7.0; the machines were 8.5 recalibrated after every 10 readings. Normal and abnormal urinalysis controls (KOVA Liqua-Trol, Hycor 8 Biomedical, Garden Grove, Calif, USA) were used to verify the accuracy of the portable and benchtop pH 7.5 meters for urine samples. The manufacturers’ reported accuracy was ±0.2 pH units for both portable models 7 and ±0.01 pH units for the reference method. The elec6.5 trodes of the portable pH meters (referred to hereafter as Chek-Mite and Checker) were placed in each urine 6 sample and a reading was taken following stabilization y = 0.973x + 0.054 of the meter. A benchtop pH meter reading immediate5.5 ly followed these measurements. After 24 hours of 5.5 6 6.5 7 7.5 8 8.5 refrigeration, samples were allowed to reach room tempH (Benchtop) perature before a secB C ond calibrated bench0.8 0.8 top pH meter reading 0.6 0.6 was taken. 0.4 0.4 Intra-assay preci0.2 0.2 sion was evaluated for 0 0 both portable models -0.2 -0.2 and the benchtop pH -0.4 -0.4 meter by repeated -0.6 -0.6 measurements, 10 -0.8 -0.8 each of normal and -1 -1 abnormal urine con-1.2 -1.2 0 5 10 15 20 trol samples. The coef5.5 6.5 7.5 8.5 ficients of variation, Frequency of Differences pH (Benchtop) calculated as the Figure 1. Comparison between dipstick and benchtop meter reference methods for measurement of pH in urine SD⫻100 divided by the samples from 40 cats. (A) Deming method comparison test. The dashed line is the line of identity (y =x). (B) NCCLS mean, were 0.9%, 0.6%, EP9-A bias plot. The solid line indicates zero bias. Dashed lines indicate the mean ± 2 SD. Mean bias = –0.12. (C) and 0.4% overall for Frequency of differences histogram. The dark line indicates the distribution of data.

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pH Difference between methods

pH Difference between methods

pH (Dipstick)

and the remainder of the urine was stored in capped 15mL polystyrene tubes (Fisherbrand, Fisher Scientific, Suwanee, Ga, USA) under refrigeration for 24 hours. Each sample of fresh urine was evaluated using pH paper (pHydrion Vivid 1-11, Micro Essential Laboratory, Brooklyn, NY, USA), urine dipstick (Multistix, Bayer Corporation, Elkhart, Ind, USA), 2 different brands of hand-held portable pH meters (Chek-Mite, Model PS30, Corning, Corning, NY, USA; Checker 1, Hanna

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example, a pH of 6 on the dipstick was comparable to a true pH of 5.81-7.18 as measured with the benchtop meter. There was close agreement between the Chek8 Mite instrument and the reference method (Figure 2). This portable pH meter was very accurate, and no con7.5 stant bias was detected. There was poor agreement 7 between the Checker portable pH meter and the reference method (Figure 3). Despite high correlation and 6.5 the least dispersion in values compared with all other tested methods, the Checker instrument showed a con6 stant bias of –0.31. The Pearson’s coefficient of correlay = 0.911x +0.592 tion was 0.99 (data not shown) for these 2 methods. Cali5.5 bration times for the 2 portable instruments differed, 5.5 6 6.5 7 7.5 8 8.5 with the Chek-Mite requiring longer to stabilize than pH (Benchtop) the Checker instrument. C B There was a signifi0.4 0.4 cant difference (P=.0001) 0.3 0.3 between the initial mea0.2 0.2 surement (0 hour) and 0.1 0.1 that recorded 24 hours 0 0 postcollection; mean ± SD -0.1 -0.1 benchtop pH meter values were 6.57± 0.54 and -0.2 -0.2 6.61± 0.53, respectively. -0.3 -0.3 However, the method -0.4 -0.4 0 5 10 15 comparison tests indicat5.5 6.5 7.5 8.5 Frequency of Differences pH (Benchtop) ed close agreement, with a bias of 0.04 (0.02-0.06, Figure 2. Comparison between Chek-Mite portable pH meter and benchtop meter reference methods for mea95% confidence interval) surement of pH in urine samples from 40 cats. (A) Deming method comparison test. The dashed line is the line (Figure 4). of identity (y = x). (B) NCCLS EP9-A bias plot. The solid line indicates zero bias. Dashed lines indicate the mean A

pH Difference between methods

pH Difference between methods

pH (Chek-Mite)

8.5

± 2 SD. Mean bias = 0.01. (C) Frequency of differences histogram. The dark line indicates the distribution of data.

the Chek-Mite, Checker, and benchtop pH meters, respectively. Using a statistical software package (Analyse-it for Microsoft Excel, Analyse-It, Leeds, UK), several evaluations were performed. The Deming method comparison test and the National Committee for Clinical Laboratory Standards (NCCLS) bias plot5 were used to assess agreement between 2 methods using the 0-hour benchtop pH meter reading as the gold standard or reference method. A paired t-test also was used to compare the mean 0-hour and 24-hour pH values as measured with the benchtop pH meter. Results Urine color appeared normal for all samples, ranging from light yellow to dark yellow or orange-yellow without evidence of blood discoloration. Descriptive data for each of the 5 methods was tabulated (Table 1).There was an average bias of –0.12 and wide disagreement at low pH values for the reagent strip method (Figure 1). For

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Discussion As expected, pH paper was the least accurate method because of the large unit scale for measurement. The reagent strip indicated a pH of 6 when the actual pH ranged from 5.81 to 7.18 with the reference method. Reagent strips could give a false sense of confidence when monitoring feline urine by suggesting that urine is acidic when it is actually neutral or slightly alkaline. Testing by dipstick was more accurate at higher pH levels, although sample numbers were fewer in this range. These findings agree with those of another study, in which urine dipsticks were less accurate than a standard laboratory pH meter.6 This inaccuracy also has been demonstrated in studies of human urine using Multistix reagent strips. In one study, considerable variation could be attributed to the technologist who performed the test.7 This difference was still present, however, when use of a semiautomated method eliminated the variation among individuals and subjective interpretations.8 Dipstick readings were falsely low at a true pH of

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)6.5 and falsely high at a true pH of *6.5.8 Portable pH meters are relatively inexpensive and could be used for home monitoring of feline urine. The 8 tight-fitting regression line and the small bias of 0.010 7.5 on the NCCLS test of agreement demonstrated that the Chek-Mite portable pH meter produced readings that 7 were closest to those of the gold standard benchtop pH meter for feline urine. The urine pH values of the Chek6.5 Mite instrument were closer to the benchtop meter 6 readings, but the actual readings took 2-3 minutes longer to stabilize compared with the Checker instru5.5 y = 1.002x – 0.317 ment. The long stabilization time was later attributed to electrode failure, which indicates that electrodes should 5 5 5.5 6 6.5 7 7.5 8 8.5 be checked and replaced as necessary, as per the manupH (Benchtop) facturer’s suggestion. Despite being the most precisely correlated method, however, the Checker instruB C ment had the poorest 0.1 0.1 agreement by the Deming 0 0 regression test and NCCLS bias plot. This -0.1 -0.1 finding indicates that lin-0.2 -0.2 ear regression tests are not the appropriate pro-0.3 -0.3 cedure for method com-0.4 -0.4 parison studies because these tests do not detect -0.5 -0.5 9 0 10 20 30 systematic errors. A type 5 6 7 8 of difference plot method Frequency of Differences pH (Benchtop) (Altman-Bland) has been Figure 3. Comparison between Checker portable pH meter and benchtop meter reference methods for measurement of pH in urine samples from 40 cats. (A) Deming method comparison test. The dashed line is the line of advocated for veterinary identity (y =x). (B) NCCLS EP9-A bias plot. The solid line indicates zero bias. Dashed lines indicate the mean ± laboratory tests when nei2SD. Mean bias=–0.31. (C) Frequency of differences histogram. The dark line indicates the distribution of data. ther method being evaluA

pH Difference between methods

pH Difference between methods

pH (Checker)

8.5

A

B

8.5

pH Difference between methods

0.2

pH (24 hrs)

8 7.5 7 6.5 6 5.5

0.15 0.1 0.05 0 -0.05 -0.1 -0.15 -0.2

5.5

6.5

7.5

8.5

5.5

pH (0 hrs)

6

6.5

7

7.5

8

8.5

pH (0 hrs)

Figure 4. Comparison of urine pH results obtained at 0 hrs and after 24 hrs of refrigeration using a benchtop pH meter. (A) Deming method comparison test. The dashed line is the line of identity (y= x). (B) NCCLS EP9-A bias plot. The solid line indicates zero bias. Mean bias = 0.04. Dashed lines indicate the mean ± 2SD.

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ated is regarded as the definitive test method; the difference between the 2 test methods is plotted against the average of the 2 methods.10 The NCCLS EP9-A bias plot method is used when one test method is regarded as the definitive test method; the difference between the 2 test methods is plotted against the reference method.5 The constant bias of –0.31 in the Checker instrument could not be explained as a calibration failure because calibration was performed before use and after every 10 samples. In some cases, systematic error may be caused by interfering substances such as proteins and lipids or to electrostatic discharges. Information provided by the company indicated that the electrodes in these portable units are not intended for long-term use, and replacement every 6 months is recommended.11 The Checker instrument had been purchased within 1 year of its use in this study. Subsequent electrode replacement improved the accuracy of the unit to within 0.1 pH unit of the reference benchtop reading, thus explaining the cause of the constant bias. Despite significant variations in pH between 0 and 24 hours of refrigeration, there was no clinical signifi-

cance attributed to the mean increase of 0.04 pH units. Thus, pet owners may safely refrigerate urine samples before transport to the clinic for measurement by a portable or benchtop pH meter. Because the vast majority of the urine samples were pH 7 or lower in this study, the effects of sample deterioration were minimal, but these effects may not be minimal in all cases. Change in pH is likely to be more notable with active sediment and infection caused by urease-containing bacteria. Measurement of feline urine pH should be evaluated with a hand-held meter rather than reagent strips, despite the higher expense over a short period of time. This approach is very important in managing a disease such as feline urolithiasis in which careful monitoring of urine pH is used to assess efficacy of treatment. Regular electrode maintenance and daily calibration of portable pH meters must be performed to ensure test accuracy. Urine reagent sticks are useful when obtaining approximations of urine pH, such as in a routine urinalysis, but should not be used when careful monitoring of urine pH is necessary. 9 ©2002 American Society for Veterinary Clinical Pathology

References 1. Vondruska JF. The effect of a rat carcass diet on the urinary pH of the cat. Companion Anim Pract. 1987;1:5-9. 2. Skoch ER, Chandler EA, Douglas GM, et al. Influence of diet on urine pH and the feline urological syndrome. J Small Anim Pract. 1991;32:413-419. 3. Buffington CA, Chew DJ. Intermittent alkaline urine in a cat fed an acidifying diet. J Am Vet Med Assoc. 1996;209:103-104. 4. Nappert G, Naylor JM. A comparison of pH determination methods in food animal practice. Can Vet J. 2001;42:364-367. 5. National Committee for Clinical Laboratory Standards. Method Comparison and Bias Estimation Using Patient Samples, Approved Guideline. Wayne, Penn: NCCLS; 1995. NCCLS EP9-A. 6. Heuter KJ, Buffington CA, Chew DJ. Agreement between two methods for measuring urine pH in cats and dogs. J Am Vet Med Assoc. 1998;213:996-998.

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7. James GP, Bee DE, Fuller JB. Accuracy and precision of urinary pH determinations using two commercially available dipsticks. Am J Clin Pathol. 1978;70:368-374. 8. Grinstead GF, Scott RE, Stevens BS, et al. The Ames Clinitek 200 Multistix 9 urinalysis method compared with manual and microscopic methods. Clin Chem. 1987;33:1660-1662. 9. Koch DD, Peters T. Selection and evaluation of methods. In: Burtis CA, Ashwood ER, eds. Tietz Fundamentals of Clinical Chemistry. 4th ed. Philadelphia, Pa: WB Saunders; 1996:170-181. 10. Jensen AL, Bantz M. Comparing laboratory tests using the difference plot method. Vet Clin Pathol. 1993;22:46-48. 11. Hanna Instruments, Inc. Temperature–resistance correlation for HANNA pH sensitive glass. Available at http://www.hannainst.com/products/electro/techover.htm. Accessed April 2, 2002.

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