Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens

Alexandra Rose Small Animal Internal Medicine Service, Justus-Liebig University, 35392 Giessen, Germany.

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Deborah Funk Small Animal Internal Medicine Service, Justus-Liebig University, 35392 Giessen, Germany.

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Reto Neiger Small Animal Internal Medicine Service, Justus-Liebig University, 35392 Giessen, Germany.

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Abstract

OBJECTIVE To compare total protein (TP) concentrations in canine pleural and abdominal fluid specimens as measured by refractometry and biuret assay.

DESIGN Diagnostic test evaluation.

SAMPLE Data regarding 92 pleural and 148 abdominal fluid specimens from dogs with various diseases.

PROCEDURES TP concentrations in fluid specimens as measured by refractometry and biuret assay were recorded. Strength of association between sets of measurements was assessed by Spearman rank correlations and Bland-Altman plots. Optimal concentration cutoff for diagnostic discrimination between exudate and nonexudate was identified by construction of receiver operating characteristic curves.

RESULTS Median TP concentration in pleural fluid specimens was 2.7 g/dL (range, 0.3 to 4.8 g/dL) for refractometry and 2.9 g/dL (range, 0.7 to 5.8 g/dL) for biuret assay. Median TP concentration in abdominal fluid specimens was 3.5 g/dL (range, 0.1 to 6.0 g/dL) for refractometry and 3.5 g/dL (range, 0.6 to 5.7 g/dL) for biuret assay. Correlation was significant between refractometric and biuret results for pleural (ρ = 0.921) and abdominal (ρ = 0.908) fluid. Bland-Altman plots revealed bias of −0.18 g/dL for pleural fluid and −0.03 g/dL for abdominal fluid for refractometry versus biuret assay. With a TP concentration of ≥ 3 g/dL used to distinguish exudate from nonexudate, sensitivity of refractometry was 77% for pleural fluid and 80% for abdominal fluid. Specificity was 100% and 94%, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE Refractometry yielded acceptable results for measurement of TP concentration in canine pleural and abdominal fluid specimens, providing a more rapid and convenient method than biuret assay. (J Am Vet Med Assoc 2016;248:789–794)

Abstract

OBJECTIVE To compare total protein (TP) concentrations in canine pleural and abdominal fluid specimens as measured by refractometry and biuret assay.

DESIGN Diagnostic test evaluation.

SAMPLE Data regarding 92 pleural and 148 abdominal fluid specimens from dogs with various diseases.

PROCEDURES TP concentrations in fluid specimens as measured by refractometry and biuret assay were recorded. Strength of association between sets of measurements was assessed by Spearman rank correlations and Bland-Altman plots. Optimal concentration cutoff for diagnostic discrimination between exudate and nonexudate was identified by construction of receiver operating characteristic curves.

RESULTS Median TP concentration in pleural fluid specimens was 2.7 g/dL (range, 0.3 to 4.8 g/dL) for refractometry and 2.9 g/dL (range, 0.7 to 5.8 g/dL) for biuret assay. Median TP concentration in abdominal fluid specimens was 3.5 g/dL (range, 0.1 to 6.0 g/dL) for refractometry and 3.5 g/dL (range, 0.6 to 5.7 g/dL) for biuret assay. Correlation was significant between refractometric and biuret results for pleural (ρ = 0.921) and abdominal (ρ = 0.908) fluid. Bland-Altman plots revealed bias of −0.18 g/dL for pleural fluid and −0.03 g/dL for abdominal fluid for refractometry versus biuret assay. With a TP concentration of ≥ 3 g/dL used to distinguish exudate from nonexudate, sensitivity of refractometry was 77% for pleural fluid and 80% for abdominal fluid. Specificity was 100% and 94%, respectively.

CONCLUSIONS AND CLINICAL RELEVANCE Refractometry yielded acceptable results for measurement of TP concentration in canine pleural and abdominal fluid specimens, providing a more rapid and convenient method than biuret assay. (J Am Vet Med Assoc 2016;248:789–794)

Pleural and abdominal effusion is common in small animal veterinary practice.1,2 Diagnostic workups for affected animals are greatly facilitated by analysis of pleural or abdominal fluid specimens. Thoracentesis and abdominal paracentesis are simple procedures by which to rapidly collect, examine, and quantify the chemical and cellular content of cavitary fluids. When coupled with consideration of clinical signs, a systematic approach to fluid analysis should help clinicians determine the cause of pleural or abdominal effusion in most situations. Tests routinely performed on pleural and abdominal fluid specimens include TP concentration measurement, cell count, and cytologic evaluation.

With refractometry, solute concentrations can be measured in various fluids. The angle of refraction for serum and plasma samples is determined by the total concentration of all solid matter. Although plasma is mostly comprised of protein, nonprotein substances routinely measured by refractometry include electrolyte, glucose, urea, and lipid concentrations.3,4 Accurate protein measurement relies on the relative consistency of these substances and is presumed to yield predictable results.5,6 Considering that, in healthy humans, serum or plasma total solids concentration is approximately 1.5 to 2 g/dL higher than the TP concentration, correct measurement of TP concentration in human serum samples necessarily involves a conversion factor.7

Since the 1960s, handheld refractometers have been used when determining protein concentration in plasma, cavitary fluids, and urine.5 Refractometric measurements of serum and plasma protein concentrations in common domestic animals have been compared with those of chemical analyzers, and results have been acceptable.8,9 However, few studies10–13 have been conducted to evaluate TP concentrations in cavitary fluid specimens and, as far as the authors are aware, only 3 reports exist on evaluation of refractometry for measurement of TP concentration in cavitary fluid specimens from dogs14,15 or cats.16

The purpose of the study reported here was to evaluate the accuracy of refractometry, compared with that of biuret assay, for measurement of TP concentration in pleural and abdominal fluids in a large sample of dogs and to assess the practical diagnostic benefit of single refractometer measurements of fluid TP concentration to easily and rapidly distinguish exudate from nonexudate.

Materials and Methods

Specimens

The medical record database of the Small Animal Clinic at the University of Giessen, Giessen, Germany was searched to identify records pertaining to pleural and abdominal fluid specimens collected from dogs with various underlying diseases between January 2008 and December 2011. To be considered for inclusion in the study, specimen records were required to include TP concentration measured by both refractometry and biuret assay. In situations in which serial assays had been performed during the same hospital stay, only data pertaining to the first fluid analysis were included.

Fluid analysis

All fluid specimens were collected into tubes without additive. Specimens were stored in a refrigerator and warmed to room temperature (approx 22°C) prior to analysis, which took place within 12 hours after specimen collection. Complete fluid analysis was performed by technicians at the Clinical Pathology Laboratory of the Small Animal Medical Service at Justus-Liebig University.

Total protein concentration in plain tubes containing pleural or abdominal fluid was measured with a clinical refractometera and biuret assay.b Monthly calibration of the clinical refractometer was performed with distilled water. Twenty microliters of each specimen was placed onto the refractometer prism by pipette, and TP concentration was estimated by comparison to a serum protein concentration scale (range, 0 to 12 g/dL). According to the manufacturer,a calibration with the serum protein concentration scale yields measurement accuracy within 0.2 g/dL. For the biuret assay, reagentc was used for determination of TP concentration. For calibration purposes, serum and plasma samples can be used.17 Limits of quantitation (0.6 g/dL) and detection (0.14 g/dL) of the assay were determined in accordance with a published protocol.18 Repeatability (within-run precision) was determined by measurement of TP concentration in 3 plasma samples of low, medium, and high concentration, and 2 control samples were assayed 20 times in accordance with the Valtec protocol.19 For calibration, a reagentd was used. For internal quality control, 2 reagentse,f were used. Each control sample was assayed daily or after a calibration. Interassay coefficient of variation of biuret assay measurements was 2%.

Statistical analysis

In all statistical tests, the biuret assay was considered the comparison method for measurement of fluid TP concentration. Because of the detection limit of the refractometer, fluid specimens containing a TP concentration of 0 g/dL on the serum protein concentration scale, as determined by the refractometer, were excluded from the study.

An ANOVA and linear regression model were used to evaluate whether the difference between refractometer and biuret assay measurements differed significantly between pleural and abdominal fluid to determine whether those measurements could be combined. When the difference was significant, results for pleural and abdominal fluid were analyzed separately.

Normality of data distribution was tested with the Shapiro-Wilk test. When data were not normally distributed, the median value was reported as measurement of central tendency. Spearman rank correlations (ρ) were calculated to determine the degree of correlation between results obtained with the 2 measurement methods. When correlations were < 0.975, Passing-Bablok regression was performed to identify presence and type of systematic error. The Wilcoxon signed rank test was performed to evaluate whether significant differences existed between paired measurements. Bland-Altman plots were created to assess the ability of the refractometer to provide accurate measurements, as indicated by the degree to which results matched those of the biuret assay. For each specimen and TP concentration measurement, bias was calculated as the refractometric concentration minus the corresponding biuret assay concentration. Mean bias and 95% (mean ± 1.96 SD) limits of agreement were calculated.

An ROC curve was plotted and the AUC calculated for each type of fluid. A pleural or abdominal fluid TP concentration of 3 g/dL was considered the cutoff to distinguish exudate from nonexudate.20–22. Sensitivity of the refractometer was defined as the probability of a test result indicating a TP concentration indicating a nonexudative (< 3 g/dL) fluid as determined by biuret assay. The specificity of the refractometer was defined as the probability of a test result indicating an exudative (≥ 3 g/dL) fluid as determined by biuret assay. Sensitivity and specificity for the combination of various refractometric cutoff values for fluid TP concentration were calculated, and the Euclidean distance between the ROC curve and upper left corner on the graph were determined. All statistical analysis was performed with statistical software.g–i Values of P ≤ 0.05 were considered significant.

Results

Records pertaining to 92 pleural and 148 abdominal fluid specimens evaluated for TP concentration by both refractometry and biuret assay were identified for inclusion in the study. The ANOVA revealed that the difference between results of refractometry and biuret assay differed significantly (P = 0.041) between pleural and abdominal fluid, and results of linear regression confirmed that difference (P = 0.024). Therefore, pleural and abdominal fluid measurements were statistically analyzed separately.

Median (range) TP concentrations in pleural and abdominal fluid specimens as measured by refractometry were 2.7 g/dL (0.3 to 4.8 g/dL) and 2.9 g/dL (0.7 to 5.8 g/dL), respectively, and by biuret assay were 3.5 g/dL (0.1 to 6.0 g/dL) and 3.5 g/dL (0.6 to 5.7 g/dL), respectively. Calculation of Spearman rank correlations revealed a moderate to high correlation between refractometric and biuret assay measurements of TP concentration in pleural fluid (ρ = 0.921; P < 0.001) and abdominal fluid (ρ = 0.908; P < 0.001) specimens, suggesting good association between results obtained with the 2 methods. Measurements had adequate agreement between methods as well; Passing-Bablok agreement equations revealed a linear relationship between the 2 methods, and analysis revealed a constant and proportional bias for pleural fluid measurements but no bias for abdominal fluid measurements (Figure 1). The Wilcoxon signed rank test revealed no significant differences between paired measurements.

Figure 1—
Figure 1—

Scatterplots of individual TP concentrations in 92 pleural fluid specimens (A) and 148 abdominal fluid specimens (B) obtained from dogs with various diseases, as measured by refractometry and biuret assay. The dashed line indicates the Passing-Bablok regression line, dotted lines indicate the 95% CI, and the solid line indicates the bisecting line.

Citation: Journal of the American Veterinary Medical Association 248, 7; 10.2460/javma.248.7.789

Bland-Altman plots revealed that the bias for each paired measurement ranged from −2.1 to 0.7 g/dL for TP concentration in pleural fluid specimens and from −2 to 1.4 g/dL for TP concentration in abdominal fluid specimens (Figure 2). For all paired measurements for the 92 pleural and 148 abdominal fluid specimens, mean ± SD difference was −0.18 ± 0.49 g/dL and −0.03 ± 0.56 g/dL, respectively.

Figure 2—
Figure 2—

Bland-Altman plots of TP concentrations in the pleural fluid specimens (A; mean ± SD bias, −0.18 ± 0.49 g/dL; 95% limits of agreement, −1.13 to 0.78 g/dL) and abdominal fluid specimens (B; mean bias, −0.03 ± 0.56 g/dL; 95% limits of agreement, −1.12 to 1.06 g/dL) in Figure 1 as measured by refractometry and biuret assay. The solid line indicates the mean difference (bias), and the dashed lines represent limits of agreement (mean ± 1.96 SD). Differences were calculated as the results for refractometry minus the results for biuret assay.

Citation: Journal of the American Veterinary Medical Association 248, 7; 10.2460/javma.248.7.789

Receiver operating characteristic curves revealed high AUCs for refractometric measurements of TP concentration in pleural fluid (AUC, 0.97; 95% CI, 0.93 to 1.0; P < 0.001) and abdominal fluid (AUC, 0.96; 95% CI, 0.93 to 0.99; P < 0.001) specimens (Figure 3). In discriminating between nonexudative and exudative pleural fluid, sensitivity and specificity of refractometry at a cutoff TP concentration of 3 g/dL were 77% (95% CI, 64.9% to 89.2%) and 100% (95% CI, 100%), respectively. In discriminating between nonexudative and exudative abdominal fluid, sensitivity and specificity at the same cutoff were 80% (95% CI, 68.7% to 91.3%) and 94% (95% CI, 89.2% to 98.8%), respectively. Sensitivity and specificity of refractometry for discrimination of nonexudate from exudate at various cutoffs for fluid TP concentration were summarized (Table 1). Cutoff values that minimized the Euclidean distance between the ROC curve and the upper left corner of the graph were 2.8 g/dL for pleural fluid and 3.2 g/dL for abdominal fluid specimens.

Figure 3—
Figure 3—

Receiver operating characteristic curves for refractometric measurement of TP concentration in the pleural fluid (A; AUC, 0.97; 95% CI, 0.93 to 1.0; P < 0.001) and abdominal fluid (B; AUC, 0.96; 95% CI, 0.93 to 0.99; P < 0.001) specimens in Figure 1. Sensitivity of the refractometer was defined as the probability of a test result indicating a nonexudative (< 3 g/dL) TP concentration as determined by the biuret assay. Specificity of the refractometer was defined as the probability of a test result indicating an exudative (≥ 3 g/dL) TP concentration as determined by the biuret assay.

Citation: Journal of the American Veterinary Medical Association 248, 7; 10.2460/javma.248.7.789

Table 1—

Sensitivity and specificity of refractometry for identification of canine pleural and abdominal fluid specimens as nonexudative at various cutoffs for TP concentration.

Cutoff (g/dL)SensitivitySpecificity
Pleural fluid
  21.0000.479
  2.150.9770.479
  2.350.9550.667
  2.550.9550.687
  2.70.9320.896
  2.90.9320.917
  30.7731.000
Abdominal fluid
  1.41.0000.360
  1.750.9900.420
  20.9900.480
  2.150.9900.520
  2.40.9800.740
  2.80.8980.820
  30.7960.940

Results were obtained by use of data pertaining to 92 pleural and 148 abdominal fluid specimens collected from dogs with various diseases.

Discussion

The present study was conducted to compare TP concentrations in pleural and abdominal fluid specimens from dogs with various diseases as measured by refractometry and biuret assay. We found a good association (ρ = 0.908 to 0.921) between measurements obtained with the 2 methods for both types of fluid. Investigators in another study15 used 2 Goldberg-type handheld refractometers to measure TP concentration in specimens of 25 body cavity fluids collected from different veterinary species and compared the results with those obtained by biuret assay, revealing a close correlation (r = 0.98) between measurement methods. In that study,15 refractometric TP concentration was determined from a conversion table and linear regression line derived from published conversion data.23,24 A second research group14 reported a small difference between refractometric and biuret assay results for analysis of canine pleural and abdominal fluid specimens, although in specimens with a TP concentration < 2 g/dL, refractometry underestimated the TP concentration. Given that this other research group14 used the same type of refractometer as in the study reported here, these underestimations might have been attributable in part to the design of the refractometer, which reportedly underestimates TP concentrations in the 0.5 to 3.0 g/dL range by approximately 0.5 g/dL.15

A third research group16 examined 26 pleural and 14 abdominal fluid specimens from cats and obtained similar results for the correlation between measurements obtained by refractometry and biuret assay (r = 0.94). In that study,16 the same refractometer was once again used as in the present study, and TP concentration was estimated by comparison to a serum protein concentration scale. Excellent correlation between methods was achieved, just as in the present study. Also similar to our findings, negative bias was identified via Bland-Altman plots for 38 of the 40 specimens, suggesting that refractometry underestimated TP concentration. However, the limits of agreement were small for both types of specimens in the present study (Figure 2), and although refractometry underestimated the fluid TP concentration, this was not a consistent finding for all specimens.

Contrary to the findings of the study15 in which Goldberg-type handheld refractometers were used, results of the present study did not suggest that the refractometer used consistently underestimated fluid TP concentration. This lack of difference could be explained by the quantity and range of measurements that we performed. Another potential source of bias is overestimation of refractometric TP concentration in certain specimens owing to the influence of other nonprotein compounds, such as cholesterol, urea, lipoproteins, glucose, and EDTA.6,25,26 Glucose would have been unlikely to have contributed to a false increase in TP concentration in the pleural and abdominal fluid specimens of the study reported here. In addition, fluid specimens were collected into tubes without any additives, so any effect of EDTA on results could be ruled out. Chylous fluids have a higher triglycerides concentration than nonchylous fluids, and triglycerides can interfere with measurement of TP concentration, but this bias can also be expected with the biuret assay.27 Therefore, it may be presumed that the direction and extent of interference from nonprotein compounds in measurement of TP concentration differed between refractometric and biuret assay in the present study.28

Traditionally, cavitary fluids can be classified by their protein concentration and cellularity as transudate, modified transudate, or exudate.20,22,29 In veterinary medicine, exudate is generally defined as cavitary fluid containing a TP concentration ≥ 3 g/dL.20–22 When a TP concentration cutoff of 3 g/dL was used in ROC curve generation in the present study, refractometry performed rather well, compared with the biuret assay, as indicated by high AUCs. When this cutoff was used, sensitivity of refractometry for classifying canine pleural and abdominal fluid as nonexudate was 77% and 80%, respectively and specificity was 100% and 94%, respectively. These findings were an acceptable range for a diagnostic test. Use of 2.5 g/dL instead of 3 g/dL as a cutoff increased the sensitivity to nearly 100% but decreased the specificity to between 67% and 74% (Table 1). Therefore, a threshold of 2.5 g/dL might be appropriate to determine whether a dog has exudative effusion. However, this conclusion is based on the presumption that a TP concentration > 3 g/dL, as measured by biuret assay, indicates an exudate. To support a refractometric classification of exudate in a single pleural or abdominal fluid specimen, analysis of fluid cellularity is warranted.

Although diagnostic accuracy, when defined as the proportion of all correct decisions (ie, proportions of all true positive and true negative results), was not directly ascertained in the present study, in human medicine a diagnostic accuracy of approximately 90% has been proposed as a minimum standard for discrimination between transudates and exudates on the basis of fluid TP concentration.30 A meta-analysis involving the diagnostic usefulness of various tests of human pleural fluid revealed that tests requiring evaluation of 1 analyte for identification of an exudate include fluid cholesterol, fluid lactate dehydrogenase, and pleural fluid protein concentration > 2.9 g/dL.31 In that study,31 the objective was to determine appropriate decision thresholds and diagnostic accuracies for pleural fluid tests that discriminate between exudative and transudative pleural fluids and evaluate the quality of the primary investigations. We are unaware of any similar studies in veterinary medicine. Studies are needed to establish the sensitivity and specificity of fluid TP concentration for correct classification of pleural or abdominal effusion.

A limitation of the present study was the retrospective design. However, complete fluid analysis was performed by personnel with sufficient training, validated methods were used, and internal and external quality assurance and control programs were routinely adhered to. Inter- and intraobserver variability in measurements made with the particular model of refractometer used in the present study has not been assessed, but results of previous studies32,33 suggest that variability associated with refractometry in general is small.

Results of the study reported here suggested that refractometry would be an acceptable, rapid, and inexpensive method for measurement of TP concentration in canine pleural and abdominal fluid specimens. For discrimination between exudative and nonexudative cavitary fluids on the basis of fluid TP concentration, use of a refractometer may be an acceptable quantitative method for practitioners. However, additional studies are needed to better define the accuracy of single refractometric measurements of fluid TP concentration in pleural or abdominal fluid specimens from dogs.

Acknowledgments

Presented in part as a poster at the 24th Annual Congress of the European College of Veterinary Internal Medicine—Companion Animals, Mainz, Germany, September 2014.

ABBREVIATIONS

AUC

Area under the curve

CI

Confidence interval

ROC

Receiver operating characteristic

TP

Total protein

Footnotes

a.

Hand refraktometer 5612, Atago Design, Euromex, Arnhem, The Netherlands.

b.

Pentra 400, ABX Horiba, Montpellier, France.

c.

Pentra Total Protein CP, ABX Horiba, Montpellier, France.

d.

Pentra Multical, ABX Horiba, Montpellier, France.

e.

Pentra N control, ABX Horiba, Montpellier, France.

f.

Pentra P control, ABX Horiba, Montpellier, France.

g.

R, version 3.1.0 beta, R Foundation for Statistical Computing, Vienna, Austria.

h.

Prism, version 6, GraphPad Software Inc, La Jolla, Calif.

i.

SPSS, version 14, SPSS Inc, Chicago, Ill.

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