Evaluation of an in-practice wet-chemistry analyzer using canine and feline serum samples

Introduction

Continuous advancement in diagnostic technology has increased both instrument reliability and the frequency of its in-house use in veterinary clinical practice. Hematology and biochemistry analyzers are the most frequently employed in first-opinion practice and have the advantages of being rapid and mostly cost-effective. Dry-chemistry analyzers are generally considered more cost-effective than wet-chemistry analyzers for low-throughput applications. For reference laboratories with high numbers of samples, large wet-chemistry analyzers offer significant savings given low reagent cost. The relatively rapid expiration of these reagents once opened precludes their use in low-throughput laboratories, however, because significant wastage would offset any savings. A smaller wet-reagent analyzer ^a is available for veterinary in-house use, although, at present, independent performance evaluation studies have not been published, to our knowledge. The aims of this study were 1) to determine the precision, total observed error (TE_obs), and acceptability of the in-practice analyzer for measuring 12 common canine and feline serum biochemical analytes and 2) to compare the results obtained from clinical samples using the in-practice analyzer to those generated from a reference commercial, high-throughput wet-chemistry analyzer. ^b

Materials and methods

Analytes

The following 12 analytes were assessed in this study: ALB, ALP, ALT, Ca, Chol, Crea, GGT, Glu, Phos, TBil, TP, and urea. The methods employed by the 2 analyzers for measurement of these analytes are shown in Table 1.

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Table 1.

Methods employed by the in-practice and reference analyzers for biochemical analysis.*

Analyte	In-practice analyzer	Reference analyzer
ALB	Bromocresol green	Bromocresol green
ALP	DGKC at 37°C, DEA	IFCC at 37°C, AMP buffer
ALT	IFCC (without P5P) at 37°C	IFCC (with P5P) at 37°C
Ca	Arsenazo	Arsenazo
Chol	GOD-PAP	Cholesterol oxidase/peroxidase colorimetric
Crea	Jaffé modified	Enzymatic colorimetric
GGT	IFCC (GLUCANA) at 37°C	IFCC (GLUCANA) at 37°C
Glu	GOD-PAP	Hexokinase
Phos	Ammonium molybdate	Ammonium molybdate
TBil	Acid diazo coupling	Acid diazo coupling
TP	Biuret modified	Biuret
Urea	Urease	Urease

*

ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein; DGKC = German Society for Clinical Chemistry (now German Society for Clinical Chemistry and Laboratory Medicine, Bonn, Germany); DEA = diethanolamine; IFCC = International Federation of Clinical Chemistry and Laboratory Medicine (Milan, Italy); AMP = 2-amino-2-methyl-1-propanol; P5P = pyridoxine-5-phosphate; GLUCANA = γ-glutamyl-3-carboxy-4-nitroanilide; GOD-PAP = Trinder oxidase-peroxidase-aminophenazone.

Results

Imprecision

All within-run CV values were <10% (Table 2). Between-run CV values were <10% except for Phos (QCM N), ALT (canine serum pool), and Ca (feline serum pool; Table 2). CV values for GGT (feline serum pool) were not reported.

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Table 2.

Within- and between-run precision data for quality control material (Eurocontrol N and Eurocontrol P; QCM N and P, respectively) and serum pools using the in-practice analyzer.*

Analyte	Within-run CV (%)					Between-run CV (%)
	QCM N	QCM P	Canine pool	Feline pool	QCM N	QCM P	Canine pool	Feline pool
ALB	0.98	1.80	1.34	2.21	2.18	1.17	2.16	2.46
ALP	2.80	1.59	3.11	2.87	2.19	1.55	1.81	5.22
ALT	8.22	2.40	3.76	4.79	2.68	1.90	11.71	4.98
Ca	1.75	1.12	1.88	0.93	1.96	1.07	4.76	10.61
Chol	1.84	1.64	1.78	2.03	1.41	2.17	0.24	1.74
Crea	1.62	0.75	3.51	1.49	5.68	4.20	2.33	1.58
GGT	2.95	2.26	7.44	NA	1.29	2.79	5.08	NA
Glu	2.43	3.08	1.48	2.22	2.23	2.31	1.66	2.12
Phos	5.07	1.77	2.96	3.19	13.72	7.21	5.39	4.09
TBil	2.91	1.13	3.51	6.77	4.51	1.30	3.17	7.28
TP	1.90	1.93	2.58	2.61	0.54	2.70	1.88	2.97
Urea	2.70	2.52	5.48	3.10	5.15	1.58	3.88	1.96

*

CV = coefficient of variation; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein; NA = not applicable (see Discussion section).

Quality requirements

All TE_obs values were below the lowest published TE_A for ALB, ALP, Chol, Crea, GGT, Glu, and TP (Table 3). Analytes for which some TE_obs values were over the lower TE_A but lower than the higher TE_A were ALT and TBil. For Ca and Phos, more than 1 TE_obs was above the higher TE_A. All σ_TEA-High were >3 except for Ca, Phos, and TP (feline serum pool only; Table 4). Many σ_TEA-Low values were also >3; TBil was the only additional analyte with a σ_TEA-Low <3 in both species.

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Table 3.

Quality control material (QCM) bias(%) and total observed error [TE_obs(%)] for the in-practice analyzer, together with published total allowable error (TE_A).*

Analyte	QCM				Serum pools				TEA†
	Bias-N	Bias-P	TEobs-N	TEobs-P	Dog		Cat
					TEobs-N	TEobs-P	TEobs-N	TEobs-P
ALB	−2.0	−9.5	6	12	6	14	7	14	15, 25
ALP	4.0	1.4	8	4	8	5	14	12	25§, 25
ALT	−7.3	2.5	13	6	31	26	17	12	25, 50
Ca	2.5	9.2	6	11	12	19‡	24‡	30‡	10, 14
Chol	−0.8	0.6	4	5	1	1	4	4	20
Crea	−6.0	−3.9	17	12	11	9	9	7	17, 20
GGT	−2.7	−7.9	5	13	13	18	NA	NA	20
Glu	−4.3	−5.1	9	10	8	8	8	9	20, 20
Phos	−27.3	14.4	55‡	29‡	38‡	25‡	35‡	23‡	15, 20
TBil	−17.5	−11.2	27	14	24	18	32	26	30||, 50
TP	2.9	4.2	4	10	7	8	9	10	10, 10
Urea	−1.8	−10.3	12	13	10	18‡	6	14	12, 16

*

N, P = Eurocontrol N and P^a, respectively; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein; NA = not applicable.

†

Published TE_A values.^4,6

‡

TE_obs > TE_A.

§

20% desirable. ⁴

||

25% desirable. ⁴

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Table 4.

Sigma (σ) values for canine and feline pooled serum measured using the in-practice analyzer.*

Analyte	Canine pooled serum		Feline pooled serum
	σTEA-Low	σTEA-High	σTEA-Low	σTEA-High
ALB	6.0	10.6	5.3	9.3
ALP	8.8	11.6	3.1	4.0
ALT	1.5	3.6	3.6	8.6
Ca	1.6	2.4	0.7	1.1
Chol	79.9	NA	11.0	NA
Crea	4.7	6.0	6.9	8.8
GGT	3.4	NA	NA	NA
Glu	9.5	NA	7.4	NA
Phos	−2.3	−1.3	−3.0	−1.8
TBil	2.4	10.2	1.0	4.5
TP	3.8	NA	2.4	NA
Urea	2.6	3.7	5.2	7.3

*

TE_A = total allowable error; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein; NA = not applicable (cannot be calculated).

Method comparison using clinical samples

Chol, Glu, and urea measurements were not significantly different between analyzers for clinical samples in either species, whereas ALB, ALP, Phos, TBil, and TP measurements were significantly different between analyzers in both canine and feline samples (Table 5). Correlations between the analyzers were <0.7 for canine Phos, feline Ca, and feline GGT (Table 6).

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Table 5.

Measurement of analytes from canine clinical samples using the in-practice and reference analyzers.*

Analyte	r	In-practice analyzer		Reference analyzer		p
		Median	Range	Median	Range
ALB (g/L)	0.91	33.5	15.9–44.9	26.8	12.8–25.8	<0.0001
ALP (IU/L)	1.00	240	25–5400	115	8–2793	0.002
ALT (IU/L)	0.97	54	8–364	53	6–447	0.800
Ca (mmol/L)	0.78	2.47	1.01–3.34	2.65	1.17–3.78	0.009
Chol (mmol/L)	0.98	5.54	1.79–15.39	5.35	1.76–17.58	0.522
Crea (µmol/L)	0.93	87.4	45.4–321.0	71.0	33.0–308.0	0.008
GGT (IU/L)	0.94	6	1–778	6	0–815	0.878
Glu (mmol/L)	0.93	4.89	0.24–19.50	5.00	0.40–18.80	0.891
Phos (mmol/L)	0.49	1.70	0.24–2.80	1.52	0.58–3.79	0.028
TBil (µmol/L)	0.70	20.0	1.2–87.0	7.2	2.0–79.2	<0.0001
TP (g/L)	0.93	64.3	28.5–83.6	59.3	27.2–80.2	0.002
Urea (mmol/L)	0.97	6.7	2.2–35.6	5.9	2.0–34.5	0.277

*

p = significance of difference between the datasets for the 2 analyzers; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein.

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Table 6.

Measurement of analytes from feline clinical samples using the in-practice and reference analyzers.*

Analyte	r	In-practice analyzer		Reference analyzer		p
		Median	Range	Median	Range
ALB (g/L)	0.71	33.7	22.3–47.3	27.4	16.8–33.6	<0.0001
ALP (IU/L)	0.93	72	3–1133	28	5–385	<0.0001
ALT (IU/L)	0.88	35	2–820	61	23–960	0.0008
Ca (mmol/L)	0.62	2.46	1.60–3.37	2.50	1.92–3.22	0.175
Chol (mmol/L)	0.77	4.17	2.31–9.20	4.00	1.52–8.76	0.673
Crea (µmol/L)	0.98	119.9	36.5–564.3	106.0	27.0–559.0	0.134
GGT (IU/L)	0.25	2	1–12	1	1–5	0.002
Glu (mmol/L)	0.86	5.35	2.59–11.63	5.40	2.60–13.20	0.880
Phos (mmol/L)	0.81	1.30	0.90–2.60	1.58	0.99–3.19	<0.0001
TBil (µmol/L)	0.86	10.1	3.5–367.0	4.4	1.9–207.0	<0.0001
TP (g/L)	0.82	71.2	47.1–91.8	67.0	42.7–87.5	0.018
Urea (mmol/L)	0.95	12.2	3.90–34 57	11.20	3.90–36.70	0.087

*

p = significance of difference between the datasets for the 2 analyzers; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein.

The most substantial bias values were seen for canine ALP (242% reference median) and TBil (218%), and for feline ALP (–307%), ALT (89%), GGT (110%), and TBil (250%; Tables 7, 8). The widest 95% LOA were observed for ALP, ALT, GGT, and TBil.

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Table 7.

Proportional error (slope), constant error (y-intercept), and bias for canine samples measured using the in-practice analyzer and relative to the reference analyzer.*

Analyte	Deming regression				Bland–Altman
	Slope	95% CI	y-intercept	95% CI	Bias	95% LOA	% in LOA
ALB (g/L)	1.23	1.11–1.36	0.43	−2.92 to 3.79	6.5	2.2 to 10.8	91
ALP (IU/L)	1.94	1.90–1.98	17.48	−4.53 to 39.49	278	−531 to 1087	95
ALT (IU/L)	0.83	0.78–0.87	8.87	2.78 to 14.95	−8	−58 to 43	91
Ca (mmol/L)	1.09	0.90–1.27	−0.40	−0.88 to 0.08	−0.18	−0.64 to 0.28	98
Chol (mmol/L)	1.01	0.96–1.06	0.22	−0.11 to 0.56	0.28	−0.83 to 1.39	95
Crea (µmol/L)	1.07	1.01–1.14	8.86	2.34 to 15.40	15.2	−9.4 to 39.8	92
GGT (IU/L)	0.98	0.96–0.99	1.20	−0.34 to 2.74	0.5	−12.4 to 13.4	97
Glu (mmol/L)	1.05	1.00–1.11	−0.20	−0.50 to 0.09	0.06	−0.94 to 1.06	100
Phos (mmol/L)	0.61	0.41–0.81	0.75	0.41 to 1.10	0.11	−0.82 to 1.05	97
TBil (µmol/L)	1.80	1.40–2.19	8.01	2.21 to 13.81	15.7	−7.1 to 38.5	95
TP (g/L)	1.19	1.11–1.28	−5.46	−10.58 to 0.33	5.6	−1.7 to 12.8	95
Urea (mmol/L)	1.07	1.03–1.11	0.21	−0.17 to 0.58	0.71	−1.19 to 2.62	98

*

CI = confidence interval; LOA = limits of agreement; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein.

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Table 8.

Proportional error (slope), constant error (y-intercept), and bias for feline samples measured using the in-practice analyzer and relative to the reference analyzer.

Analyte	Deming regression				Bland–Altman
	Slope	95% CI	y-intercept	95% CI	Bias	95% LOA	% in LOA
ALB (g/L)	1.51	1.11–1.90	−7.28	−17.90 to 3.35	6.3	−0.2 to 12.9	90
ALP (IU/L)	2.63	2.50–2.76	−3.95	−15.94 to 8.05	86	−151 to 323	97
ALT (IU/L)	0.66	0.59–0.74	−4.27	−23.56 to 15.03	−54	−247 to 139	97
Ca (mmol/L)	1.62	1.07–2.17	−1.63	−3.02 to −0.24	−0.08	−0.65 to 0.49	97
Chol (mmol/L)	1.14	0.93–1.34	−0.41	−1.30 to 0.47	0.15	−1.38 to 1.67	95
Crea (µmol/L)	1.02	0.99–1.06	12.37	6.64 to 18.10	15.5	−9.1 to 40.0	93
GGT (IU/L)	5.24	1.16–9.32	−5.94	−13.96 to 2.08	1.1	−3.1 to 5.3	98
Glu (mmol/L)	1.01	0.89–1.13	0.00	−0.73 to 0.74	0.08	−1.40 to 1.55	97
Phos (mmol/L)	0.80	0.70–0.91	0.07	−0.11 to 0.24	−0.26	−0.66 to 0.15	93
TBil (µmol/L)	1.62	1.53–1.72	2.07	−1.31 to 5.46	11.0	−31.3 to 53.2	98
TP (g/L)	1.02	0.83–1.21	2.70	−10.08 to 15.49	4.0	−6.6 to 14.5	97
Urea (mmol/L)	1.07	0.99–1.15	0.67	−0.40 to 1.75	1.48	−2.15 to 5.11	98

*

CI = confidence interval; LOA = limits of agreement; ALB = albumin; ALP = alkaline phosphatase; ALT = alanine aminotransferase; Ca = calcium; Chol = cholesterol; Crea = creatinine; GGT = gamma-glutamyl transferase; Glu = glucose; Phos = phosphate; TBil = total bilirubin; TP = total protein.

Discussion

Large-scale wet-chemistry analyzers are employed by most veterinary diagnostic laboratories because of their speed, overall reliability, and consistency of results. The costs of running these analyzers can be prohibitive for general practitioners in first-opinion practice, however, especially when throughput is relatively low. Validation of smaller, less expensive analyzers is therefore of most benefit to practices who desire wet-chemistry analysis without the incumbent costs.

The results of the repeatability study show that the within-run CV values for both QCM and both serum pools were mostly ≤5% (42/47 values), and all were <9%. The majority of between-run CV values (38/47) were also ≤5%, with all values <14%. The in-practice analyzer therefore has high precision for measurement of most analytes in dogs and cats, and moderate precision for the remaining analytes.

The between-cat variation in GGT in our study, even with the inclusion of cats with apparent cholestasis (based on ALP and TBil values), was relatively very low compared with that for the dog. Our maximum observed feline GGT activity measured by the in-practice analyzer was 12 IU/L, although TBil and ALP were normal in this cat. The maximum reference analyzer GGT activity was 5 IU/L (in-practice analyzer: 4 IU/L); ALP and TBil were both markedly increased in this sample. In contrast, the maximum canine GGT measured was 778 IU/L. In our experience, even cats with marked cholestasis are observed to have substantially lower GGT levels than other species, and milder increases following extrahepatic bile duct obstruction are seen in the cat than in the dog. ⁷ With such narrow between-cat variation, GGT values need to be reported to at least 1 decimal place for meaningful repeatability analysis because each 1 IU/L represents ≥8% of the maximum value. GGT is measured to zero decimal places using the in-practice analyzer, however, which generated very large and meaningless CV values. The results were therefore excluded from the study, and dependent calculations (feline GGT TE_obs and σ values) were not performed. This issue has been encountered in other similar studies, and results were likewise excluded. ³

QCM TE_obs-N(%) and TE_obs-P(%) were broadly similar for all analytes, and, with the exception of canine ALT, were also broadly similar to the pooled samples. Seven of the analytes (ALB, ALP, Chol, Crea, GGT, Glu, and TP) had all TE_obs(%) values below the lower published TE_A, which demonstrates acceptability based on observed error for these methods. TE_obs(%) for TBil and ALT were lower than the higher TE_A, which also suggests acceptability for these methods. TE_obs-P(%) for canine urea was the only TE_obs value for this analyte above TE_A, but the increase was small (2%) and likely to be of little significance. Several TE_obs(%) for Ca and all TE_obs(%) for Phos were well above the TE_A, which suggests the in-practice analyzer methods are not acceptable for measuring these analytes based on observed error.

The computed σ metric value for an assay and its graphical representation, the MEDx chart, are performance indicators used to show assay reliability. ⁵ This information complements allowable error analysis and ensures that the minimum desired quality standards for an assay are met. In addition, these analyses are used to determine the stringency of quality control rules for that particular assay, with lower values requiring a greater number of, and more stringent, rules to ensure error detection. The highest σ values in our study were generally observed for analytes with TE_obs < TE_A, which is in part caused by low CV and/or bias, and relatively high TE_A. Canine analytes with σ_TEA-Low values >4 (good performance) were ALB, ALP, Chol, Crea, and Glu, with TBil σ_TEA-High >4. ALT, GGT, TP, and urea were all >3 for σ_TEA-Low and/or σ_TEA-High, which suggests that the performance for measuring these analytes is likely to be sufficient but with room for improvement. For feline samples, analytes with σ_TEA-Low >4 were ALB, Chol, Crea, Glu, and urea, with σ_TEA-High >4 for ALP, ALT, and TBil. Ca and Phos performance in both species, and TP performance in the cat, was poor or worse. No single factor was identified to explain the low feline TP σ value, as bias and CV were both low. A relatively low TE_A of 10% (much lower than ALB, for example) appears contributory, however, and a TE_A of 12% would move the σ value to >3.

Ca performance is likely to be affected, at least in part, by the relatively low TE_A compared with most other analytes. A low TE_A is expected for analytes that require tight biological control, and so the performance requirement is high to ensure that small deviations are detected accurately and reliably. Ca bias was 2% for QCM N, with between-run CV of 5% and 10% in the dog and cat, respectively. This suggests that improvement in precision is required to increase the σ value.

High TE_obs(%) and very poor σ values for Phos measurement appear to be the result of high bias(%): QCM N generated a bias of –27%, and QCM P a bias of 14%, despite controls falling within the recommended ranges given in the technical inserts. This suggests there may have been a failure in calibration. Recalibration of the instrument with 2 different batches of QCM N and P did not affect the Phos measurements of the QCM (not shown). Bias values for the other analytes were a mixture of positive and negative, and of relatively small magnitude for most analytes, which excludes errors in reconstitution of 1 or both of the QCM. Failure of calibration is therefore potentially the result of either incorrect concentration of Phos in both QCMs (which is unlikely given that bias was large for both QCM but in different directions) or a technical fault in the assay.

Correlation between the analyzers was mostly very high for canine samples, with Ca showing moderate correlation and Phos showing poor correlation. The datasets for many canine analytes were significantly different between the analyzers, however, and the Deming regression analyses revealed that this was mostly because of constant error in the measurement of the analytes by the in-practice analyzer relative to the reference analyzer. Correlations were also high or very high for feline samples, with moderate correlation seen for ALB and Chol, and poor for Ca and GGT. Major differences between the analyzer datasets also appeared to be the result of constant rather than proportional error with the exception of GGT.

Agreement between the analyzers was assessed from the results of the Bland–Altman analysis. For many of the analytes, ≥95% of results fell within the 95% LOA; however, the bias was too large and/or 95% LOA was too wide to be meaningful. ¹ Canine Glu and TP, and feline TP, had a small mean bias and relatively narrow LOA, as well as ≥95% results within the 95% LOA, consistent with good agreement. Agreement for the remaining analytes was considered unacceptable.

Our study had some limitations. Ideally, precision, TE_obs, and σ metrics are calculated for 2 or 3 different analyte levels (low, within-reference, and high) to show performance over the range of clinical samples. In our study, we determined 1 precision, TE_obs, and σ metric value for each analyte given the use of a single pooled sample for each species. It must also be noted that veterinary TE_A values are generally based on results from canine studies, and all TE_A values in this study were defined for dogs. It is therefore possible that these values are not always appropriate for cats when reference intervals differ significantly between species. Complete validation of a method should include reportable range, recovery, and interference measurements. For simplicity, these were not performed during this study, and it is recommended that these be determined prior to clinical use. Last, duplicate measurement of analytes in the clinical samples may have improved agreement between the analyzers. Were the interchangeability of reference intervals between the analyzers of critical importance, this would have been preferred; for the purposes of this study and other studies, it was not necessary. ³