Determination of reference intervals for plasma biochemical values in clinically normal adult domestic shorthair cats by use of a dry-slide biochemical analyzer

Brice S. Reynolds Department of Clinical Sciences, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.

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Karine G. Boudet Department of Clinical Sciences, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.
Unité Mixte de Recherche 181 Physiopathologie et Toxicologie expérimentales, Institut National de la Recherche Agronomique, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.

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Claude A. Germain Department of Clinical Sciences, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.
Unité Mixte de Recherche 181 Physiopathologie et Toxicologie expérimentales, Institut National de la Recherche Agronomique, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.

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Jean-Pierre D. Braun Department of Clinical Sciences, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.
Unité Mixte de Recherche 181 Physiopathologie et Toxicologie expérimentales, Institut National de la Recherche Agronomique, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.

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Herve P. Lefebvre Department of Clinical Sciences, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.
Unité Mixte de Recherche 181 Physiopathologie et Toxicologie expérimentales, Institut National de la Recherche Agronomique, National Veterinary School of Toulouse, 23, chemin des Capelles, BP 87614, 31076 Toulouse cedex 03, France.

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Abstract

Objective—To establish reference intervals of plasma biochemical values in healthy adult domestic shorthair (DSH) cats by use of controlled conditions.

Animals—95 healthy client-owned cats.

Procedures—Food was withheld from the cats overnight. All blood samples were obtained on the same day, at the same location, and by the same investigator. Blood samples were collected from a cephalic vein into lithium heparin tubes. After centrifugation of blood samples, plasma supernatants were harvested and stored at −20°C until assayed for total proteins, albumin, creatinine, urea, glucose, calcium, phosphates, sodium, chloride, potassium, and CO2 concentrations and alkaline phosphatase and alanine aminotransferase activities.

Results—Reference intervals in healthy adult DSH cats were 65 to 85 g/L for total proteins, 27 to 39 g/L for albumin, 89 to 207 μmol/L for creatinine, 6.6 to 11.3 mmol/L for urea, 4.1 to 8.2 mmol/L for glucose, 2.4 to 2.9 mmol/L for calcium, 1.1 to 2.1 mmol/L for phosphates, 153 to 161 mmol/L for sodium, 120 to 127 mmol/L for chloride, 3.3 to 4.2 mmol/L for potassium, 15 to 21 mmol/L for CO2, 32 to 147 U/L for alkaline phosphatase, and 34 to 123 U/L for alanine aminotransferase.

Conclusions and Clinical Relevance—This study provided reference intervals for plasma analytes in adult DSH cats. The influence of potential confounding factors was minimized through use of controlled preanalytic and analytic conditions. However, these results cannot be extrapolated to other feline breeds or used to interpret results from other biochemical analyzers.

Abstract

Objective—To establish reference intervals of plasma biochemical values in healthy adult domestic shorthair (DSH) cats by use of controlled conditions.

Animals—95 healthy client-owned cats.

Procedures—Food was withheld from the cats overnight. All blood samples were obtained on the same day, at the same location, and by the same investigator. Blood samples were collected from a cephalic vein into lithium heparin tubes. After centrifugation of blood samples, plasma supernatants were harvested and stored at −20°C until assayed for total proteins, albumin, creatinine, urea, glucose, calcium, phosphates, sodium, chloride, potassium, and CO2 concentrations and alkaline phosphatase and alanine aminotransferase activities.

Results—Reference intervals in healthy adult DSH cats were 65 to 85 g/L for total proteins, 27 to 39 g/L for albumin, 89 to 207 μmol/L for creatinine, 6.6 to 11.3 mmol/L for urea, 4.1 to 8.2 mmol/L for glucose, 2.4 to 2.9 mmol/L for calcium, 1.1 to 2.1 mmol/L for phosphates, 153 to 161 mmol/L for sodium, 120 to 127 mmol/L for chloride, 3.3 to 4.2 mmol/L for potassium, 15 to 21 mmol/L for CO2, 32 to 147 U/L for alkaline phosphatase, and 34 to 123 U/L for alanine aminotransferase.

Conclusions and Clinical Relevance—This study provided reference intervals for plasma analytes in adult DSH cats. The influence of potential confounding factors was minimized through use of controlled preanalytic and analytic conditions. However, these results cannot be extrapolated to other feline breeds or used to interpret results from other biochemical analyzers.

Plasma biochemical analysis has become a routine part of clinical evaluation in small animal medicine.1 In general, the values obtained are interpreted by comparison with a reference interval. A major issue concerning this approach is that, to our knowledge, there are few publications that describe the systematic determination of reference intervals in healthy cats. Currently used reference intervals often come from textbooks or are provided by analyzer manufacturers. In many cases, the reference population is not indicated or is poorly defined, and factors that may have affected the results are not always specified.1,2 The objective of the study reported here was to establish reference intervals for plasma analytes determined by use of a dryslide biochemical analyzer for a representative sample of DSH cats. Effects of age, sex, body weight, and housing condition (ie, indoor vs outdoor) on analyte values were also assessed.

Materials and Methods

Sample population—Client-owned cats from a first-opinion private practice were used in the study; owners were invited to participate via telephone. Blood samples were obtained from healthy DSH cats at a veterinary clinic.a None of the cats were receiving treatment or had a known medical condition, and all were considered healthy on the basis of medical history, results of the most recent annual examination performed at the clinic, and lack of clinical signs of illness on the day of blood collection. Cats were excluded from the study on the basis of breed, specific physiologic conditions (pregnancy or lactation), clinical signs or a history of any pathologic condition, or any drug treatments during the 4 weeks before sample collection.

The protocol for the study was designed in accordance with standard guidelines3 for determining reference intervals for quantitative clinical laboratory tests. This study was conducted in accordance with conditions approved by the French Ministry of Agriculture and adhered to published guidelines.4 Written consent was obtained from all owners of participating cats.

Sampling procedure—Appointments were made with all participating owners to facilitate the collection of samples from all cats on the same day. Owners were instructed to withhold food from their cats during the period 12 to 24 hours immediately preceding blood collection. Each cat and its owner remained in the waiting room for a brief period after arrival at the clinic. Blood samples were obtained within 20 minutes after arrival. Each cat was gently restrained in a sitting position in the presence of the owner; the same trained handler restrained each cat. One investigator collected all blood samples. Blood was obtained from a cephalic vein with a 25-gauge needle and two 200-μL capillary tubes that contained heparinate lithium,b as described elsewhere.5 The hair was not clipped before venipuncture, but the puncture site was prepared by swabbing with 70% alcohol. Total time to fill the 2 tubes was < 1.5 min/cat.

Sample processing—Blood samples were centrifuged (3,000 × g for 10 minutes) within 30 minutes after collection, and the plasma supernatant was harvested and then stored at −20°C. Plasma assays were performed on days 4 and 5 after blood samples were collected. On the days of testing, plasma samples were allowed to thaw at 22°C for 30 minutes before being assayed.

Assays—Plasma biochemical assays were performed by use of a dry-slide biochemical analyzer.c Plasma analytes tested were total proteins, albumin, creatinine, urea, ALT, ALP, glucose, calcium, phosphates, sodium, chloride, potassium, and CO2. Quality control was based on weekly measurement of control solutions with known concentrations.d According to the manufacturer, values for the assays had been determined by use of standard reference materials available from the National Institute of Standards and Technology. Repeatability of each assay was estimated by use of 10 consecutive replicates of each analysis of the same batch of control solutions. Within-laboratory imprecision was estimated by use of weekly, single measurements of each analyte of the same batch of control solutions for a period of 10 consecutive weeks.

Stability of analytes during storage at −20°C—A stability experiment was performed to assess potential alteration of analytes as a result of freezing of plasma samples, storage at −20°C, and subsequent thawing. Blood samples were obtained from 14 DSH cats and processed as described previously. The plasma was divided into 2 aliquots. The first aliquot was immediately assayed for the aforementioned analytes. The second aliquot was stored at −20°C for 5 days, and then it was allowed to thaw at 22°C for 30 minutes and assayed. Differences between results for fresh plasma and plasma stored at −20°C were calculated.

Table 1—Repeatability and within-laboratory imprecision estimates for 2 control solutions of plasma analytes measured by use of a dry-slide biochemical analyzer.

RepeatabilityWithin-laboratory imprecision  
Analyte AAnalytic rangeSolutionValueSDCV(%)SDCV(%)
Total proteins (g/L)20–1101431.22.80.61.5
2681.92.71.01.5
Albumin (g/L)10–601250.41.50.62.5
2430.51.10.71.6
Creatinine ($mUmol/L)4–1,2381870.90.93.03.1
25233.50.78.11.6
Urea (mmol/L)0.7–42.815.60.091.360.081.16
216.40.190.940.140.69
ALT(U/L)3–1,0001370.92.11.63.7
22041.40.83.31.8
ALP(U/L)20–1,5001741.61.51.81.8
24695.81.013.62.5
Glucose (mmol/L)1.1–34.714.80.030.700.173.75
215.90.070.460.231.41
Calcium (mmol/L)0.25–3.4912.300.0210.8990.0321.341
23.000.0140.4480.0331.041
Phosphates (mmol/L)0.16–4.2011.060.0121.0970.0171.595
22.410.0100.4510.0150.626
Sodium (mmol/L)75–25011240.70.62.72.3
21470.80.52.11.5
Chloride (mmol/L)50–1751860.50.71.51.8
21180.60.62.72.5
Potassium (mmol/L)1–14130.00.70.01.4
25.60.00.80.12.0
CO2 (mmol/L)5–401130.62.50.73.0
2270.75.30.74.8

CV = Coefficient of variation.

Figure 1—
Figure 1—

Gaussian distribution of plasma creatinine concentrations measured in samples obtained from 92 healthy DSH cats. The corresponding reference interval was calculated as mean ± 2 SD.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.471

Statistical analysis—Reference intervals were defined as the central 95% interval bounded by the 2.5th and 97.5th percentiles. Statistical analyses were performed by use of a statistical software package.e Data were first tested for normality by use of the Kolmogorov-Smirnov test. A value of P < 0.05 was considered significant. When the data distribution was Gaussian, 95% reference intervals were calculated as mean ± 2 SD. When the data were normally distributed after mathematic (ie, logarithmic, square root, or inverse) transformation, the 2.5th and 97.5th percentiles were calculated as mean ± 2 SD in the transformed data scale, and these percentiles were subsequently converted to the original data scale by use of the inverse function. When the data distribution remained non-Gaussian after mathematic transformation, a nonparametric approach was performed (ie, values between the 2.5th and 97.5th percentiles of the distribution were used to determine the reference intervals). The reference intervals obtained were subsequently compared with those published in 3 veterinary textbooks.2,6,7

Effects of age, sex, body weight, and housing condition on each plasma analyte value were evaluated by use of the following general linear model8:

article image
where Yi,j,k is the observed value for cat k (k = 1 to n) with sex i and housing condition j; μ is the general mean effect; sexi is the effect of sex (i = male or female); housing conditionj is the effect of housing condition (j = indoor or outdoor); a and b are the coefficients of the terms age and body weight, respectively; agei,j,k is the age of cat k with sex i and housing condition j; body weighti,j,k is the body weight of cat k with sex i and housing condition j; and Ei, j, k is the residual term of the model. A value of P < 0.05 was considered significant.

Figure 2—
Figure 2—

Distribution of plasma urea concentrations measured in samples obtained from 88 healthy DSH cats. Nontransformed data had a non-Gaussian distribution (A), whereas logarithmic transformation resulted in a Gaussian distribution (B). The corresponding data were used to determine the 2.5th and 97.5th percentiles as mean ± 2 SD in the transformed data scale, and these percentiles were subsequently converted to the original data scale by use of the inverse function.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.471

Results

Blood samples were collected from 95 DSH cats. Mean ± SD age and body weight of the cats were 7 ± 5 years (median, 6 years; range, 0.6 to 20 years) and 4.6 ± 1.1 kg (median, 4.5 kg; range. 2.3 to 7.8 kg), respectively. Of the 95 cats, 86 (90.5%) were neutered (48 spayed females and 38 castrated males); 7 were sexually intact males, and 2 were sexually intact females. Sixty (63.2%) cats were housed strictly indoors.

Coefficients of variation for repeatability and within-laboratory imprecision of measurements obtained by use of the dry-slide biochemical analyzer were < 4.8% for control solutions of all analytes evaluated in our study (Table 1). Results could not be obtained for all 13 plasma analytes in all cats because the blood volume collected (≤ 400 μL) was not always sufficient to yield the amount of plasma needed to perform all the assays. No plasma samples had evidence of hemolysis. Distributions of plasma concentrations did not differ from a Gaussian distribution for total proteins, albumin, and creatinine (Figure 1). Distributions of plasma urea concentrations and ALP activities after logarithmic transformation and of glucose concentrations after inverse transformation did not differ from a Gaussian distribution (Figure 2). The corresponding reference intervals were calculated by use of a parametric method (Table 2). Distributions of plasma concentrations of phosphates, calcium, sodium, chloride, potassium, and CO2 and plasma activity of ALT could not be transformed into a Gaussian distribution (Figure 3). The corresponding reference intervals were determined by use of a nonparametric method. Comparison of the reference intervals obtained in our study with those published in veterinary textbooks revealed some similarities, discrepancies, and partial overlaps between sources (Table 3).

Table 2—Reference intervals for plasma analytes in healthy DSH cats.

AnalyteNo. of catsDistributionMeanMedianSDRangeReference interval
Total proteins (g/L)95G7575563–9265–85
Albumin (g/L)84G3333325–4027–39
Creatinine ($mUmol/L)92G1481492952–21389–207
Urea (mmol/L)88GL9.18.81.95.7–18.36.6–11.3
ALT(U/L)70NP60542315–12934–123
ALP(U/L)71GL66622530–17432–147
Glucose (mmol/L)94GI5.75.41.14.1–10.94.1–8.2
Calcium (mmol/L)82NP2.62.60.12.4–3.12.4–2.9
Phosphates (mmol/L)83NP1.61.60.31.1–2.71.1–2.1
Sodium (mmol/L)94NP1571572152–166153–161
Chloride (mmol/L)72NP1241242120–130120–127
Potassium (mmol/L)92NP3.83.70.33.0–4.53.3–4.2
CO2 (mmol/L)88NP1817214–2415–21

G = Gaussian. GL = Gaussian distribution after logarithmic transformation. GI = Gaussian distribution after inverse transformation. NP = Nonparametric.

Figure 3—
Figure 3—

Non-Gaussian distribution of plasma concentrations of phosphates measured in samples obtained from 83 healthy DSH cats. The reference interval was determined by use of a nonparametric method.

Citation: American Journal of Veterinary Research 69, 4; 10.2460/ajvr.69.4.471

No outliers were detected for plasma concentrations of total proteins and potassium, and 1 outlier was detected for each of the other analytes, with the exception of ALP activity, for which 2 cats had values that were inconsistent with those of the other cats. However, no abnormality concerning medical history or clinical examination could be found to warrant discarding these values.

Plasma glucose concentration increased significantly with age of the cats (P = 0.010), whereas plasma concentrations of phosphates (P < 0.001) and albumin (P = 0.002) decreased significantly with age (Table 4). Plasma CO2 concentration was significantly (P = 0.018) higher in males than in females (19 ± 1.2 mmol/L vs 16.4 ± 0.9 mmol/L, respectively). Plasma concentrations of glucose (P < 0.001), total proteins (P < 0.001), albumin (P = 0.003), and creatinine (P = 0.048) increased with body weight (Table 5). Plasma concentrations of phosphates (P = 0.016) and CO2 (P = 0.012) and plasma ALP activity (P = 0.033) were significantly higher in indoor cats, whereas plasma chloride concentration was significantly (P = 0.032) lower (Table 6). The stability experiment revealed that storage of plasma samples at −20°C did not cause any alterations of values for any of the tested analytes (Table 7).

Discussion

Most of the reference intervals commonly used in cats and other animal species have been poorly defined because of lack of information about the reference population (especially the inclusion and exclusion criteria), limited number of animals tested, lack of controlled preanalytic and analytic conditions, or inadequate statistical analysis. It is generally recommended, but unrealistic, that each laboratory establish its own reference intervals.3 One of the major reasons cited for the lack of established reference intervals for each laboratory in veterinary and human medicine is that samples intended for use by reference laboratories are hard to obtain from healthy subjects because samples are often collected to make or confirm a clinical diagnosis. Discrepancies between the reference intervals established in the study reported here and those published in textbooks may be attributable to differences in the tested population and analytic methods, but the exact reason cannot be determined because most textbooks do not provide any information about how values were obtained. Furthermore, some textbooks do not indicate the number of animals used to establish the reference intervals.6,7 Moreover, it must be emphasized that reference intervals are dependent on the instruments and methods used.

Table 3—Comparison of the reference intervals obtained for plasma analytes measured in samples obtained from 95 healthy cats reported here and those published in veterinary textbooks.

Analyte95 DSH catsTextbook 12Textbook 26Textbook 37
Total proteins (g/L)65–8553–8554–7852–79
Albumin (g/L)27–3924–3521–40
Creatinine (μmUmol/L)89–20760–16371–15988–177
Urea (mmol/L)6.6–11.34.8–11.63.3–5.0
ALT (U/L)34–12320–856–8310–150
ALP (U/L)32–14713–11625–9310–200
Glucose (mmol/L)4.1–8.22.8–8.43.9–6.13.9–6.4
Calcium (mmol/L)2.4–2.92.2–3.01.6–2.61.8–3.0
Phosphates (mmol/L)1.1–2.11.2–3.01.5–2.61.0–1.6
Sodium (mmol/L)153–161147–161147–156145–155
Chloride (mmol/L)120–127115–125117–123111–126
Potassium (mmol/L)3.3–4.24.2–6.14.0–4.54.3–5.2
CO2 (mmol/L)15–2110–2117–2417–28

— = Value not provided.

Table 4—Mean ± SD concentrations of plasma glucose, phosphates, and albumin in healthy DSH cats, by age classification.

Age categories*  
Analyte< 4 years4 to 9 years> 9 years
Glucose (mmol/L)5.3 ± 0.6 (31)5.6 ± 1.0 (31)6.1 ± 1.5(32)
Phosphates (mmol/L)1.7 ± 0.3(27)1.6 ± 0.3(24)1.5 ± 0.2 (30)
Albumin (g/L)34 ± 3 (27)34 ± 3 (26)32 ± 3 (31)

Number of cats in each category is indicated in parentheses.

*Data have been classified into arbitrary age categories (young, < 4years; adult, 4to 9 years; and senior, > 9 years) for the purpose of reporting results.

Table 5—Mean ± SD concentrations of plasma glucose, total proteins, albumin, and creatinine in healthy DSH cats, by body weight classification.

Body weight category*  
Analyte<4kg4 to 5 kg>5kg
Total proteins (g/L)75 ± 6 (26)74 ± 4 (38)78 ± 5 (31)
Albumin (g/L)32 ± 3 (22)33 ± 3 (34)34 ± 3 (28)
Creatinine ($mUmol/L)142 ± 36 (25)144 ± 31 (37)161 ± 22(30)
Glucose (mmol/L)5.4 ± 0.7 (26)5.4 ± 0.8 (38)6.3 ± 1.5(30)

Number of cats in each category is indicated in parentheses.

*Data have been classified into arbitrary categories of body weight for the purpose of reporting results.

Table 6—Mean ± SD values of plasma analytes that differed significantly between indoor and outdoor healthy DSH cats.

AnalyteIndoor catsOutdoor catsP*
ALP (U/L)71 ± 29(42)57 ± 14(29)0.033
Phosphates (mmol/L)1.64 ± 0.28(51)1.52 ± 0.26(32)0.016
Chloride (mmol/L)124 ± 2 (43)125 ± 2 (29)0.032
CO2 (mmol/L)18 ± 2 (54)17 ± 1 (34)0.012

Number of cats in each category is indicated in parentheses.

*Values differed significantly at P< 0.05.

Table 7—Stability of plasma analytes after storage at −20°C for 5 days, measured in samples obtained from 14 DSH cats.

AnalyteFresh* (Mean ± SD)Frozen (Mean ± SD)Median (range) of difference (%)†
Total proteins (g/L)77 ± 475 ± 53.3 (−3.0 to 7.1)
Albumin (g/L)34 ± 335 ± 32.8 (−10.9 to 4.4)
Creatinine (μmUmol/L)122 ± 22122 ± 220.8 (−2.4 to 4.0)
Urea (mmol/L)8.8 ± 1.58.8 ± 1.51.0 (−2.6 to 2.9)
ALT (U/L)47 ± 848 ± 83.6 (−9.8 to 5.3)
ALP (U/L)51 ± 2052 ± 214.2 (−8.1 to 10.5)
Glucose (mmol/L)5.3 ± 0.45.4 ± 0.42.1 (−3.6 to 3.0)
Calcium (mmol/L)2.6 ± 0.12.6 ± 0.11.2 (−2.0 to 3.3)
Phosphates (mmol/L)1.8 ± 0.31.8 ± 0.31.4 (0.0 to 3.8)
Sodium (mmol/L)155 ± 1.7156 ± 1.20.6 (−1.3 to 0.6)
Chloride (mmol/L)122 ± 3123 ± 30.0 (−0.9 to 1.6)
Potassium (mmol/L)4.0 ± 0.64.0 ± 0.60.0 (−3.3 to 0.0)
CO2 (mmol/L)22 ± 221 ± 22.1 (0.0 to 13.6)

*Values for fresh plasma correspond to the values obtained by assaying plasma immediately after samples were obtained. †The maximal difference corresponds to the difference between values obtained for frozen plasma and fresh plasma for a particular cat.

Ideally, reference intervals should be determined through an appropriately designed study conducted under controlled conditions. The first step in establishing population-based reference values is to select a reference sample group that is representative of the target population.9–11 Only DSH cats were used in the study reported here because this breed was deemed most representative of the general feline population. Breed-dependent effects on reference intervals in cats cannot be excluded because values of certain analytes can vary by breed. For example, plasma creatinine concentrations are often higher in Birman cats than in cats of other breeds.12 All the cats in our study were client-owned animals and believed to be representative of the population of interest. As recommended by the guidelines3 of the Clinical and Laboratory Standards Institute, cats were not ill and did not require hospitalization or treatment. One important step, as for any study of reference intervals, was to define the criteria for health. In the study reported here, cats were considered healthy on the basis of a lack of clinical signs on the day of sample collection and no recent history of medication. The medical history could also be verified because all cats received a physical examination on a regularly scheduled basis. Only cats from which food was withheld were used, with the duration of food withholding ranging from 12 to 24 hours. This duration is typical for adult feline surgical patients.13 Because blood samples were collected throughout the day, a fixed period for withholding of food was not possible. Withholding of food was deemed necessary because plasma analyte concentrations may reflect postprandial changes14 and because large within-animal fluctuations may be evident after a meal, depending on the time of sample collection.

The study was designed so that the conditions of the visit to the veterinary clinic were similar to those encountered in routine clinical practice. Blood samples were collected from all cats in the same manner, by the same veterinarian, and within a single day. The method of blood collection was selected because, in our experience, it can be quickly and easily performed, requires only light restraint, and is tolerated well by most cats. Data obtained through the use of the capillary tube technique can be extrapolated with confidence to conditions in which blood samples are collected with vacuum tube systems, at least for plasma glucose, creatinine, and potassium concentrations and ALT and ALP activities.5

Techniques for the collection of blood samples, sites of venipuncture, and extent and degree of restraint during the procedure may also affect results of biochemical analyses.1,15,16 For example, plasma activities of ALP in cats differ between blood samples obtained from the cephalic and jugular veins.1 Struggling associated with collection of blood samples may result in substantial increases in plasma glucose and lactate concentrations.16 Obtaining a clean and direct venous blood sample from a cat can be difficult because of the natural reluctance of cats to be physically restrained. Biochemical assay results may be altered by contamination of the specimen with substrates or enzymes from tissue surrounding a vein.9 For example, falsely high results for plasma activity of creatine kinase have been obtained for dogs and horses as a result of incorrect venipuncture.17 Plasma concentration of glucose in 8 cats tested in the study reported here was > 7 mmol/L, which could be interpreted as abnormally high; this was attributed to stress.18,19 Because stress-induced hyperglycemia is expected for similar conditions in practice settings and should not be misinterpreted as a pathologic finding, all values were included and taken into account in determining the reference interval for plasma glucose concentration. The upper limit of our reference interval for plasma glucose concentration is similar to that proposed in another study.18

A delay in separating plasma from blood cells can lead to leakage of some intracellular constituents and cellular consumption of some plasma constituents (primarily glucose).9 Processing artifacts were therefore minimized by centrifugation of all blood samples within 30 minutes after collection and immediate storage of the plasma supernatant at −20°C for no more than 5 days until assayed. Although such a storage procedure may have altered concentrations of the plasma constituents, the stability of analytes during storage and thawing of deep-frozen plasma was excellent for the conditions used here.

Reference intervals can also be influenced by assay techniques.9–11 Because a specific analyzer was used in our study, other investigators who wish to apply the reference intervals reported here to their own laboratories will need to address certain issues. First, their laboratory population should be similar to ours. Then, reference intervals for the analytes should be developed in accordance with standard rules for method comparison and bias estimation.

In our study, the final size of the reference sample varied between 70 and 95 cats, depending on the plasma analyte assessed; this is similar to the size of the reference samples in other studies1,18–27 of cats. A minimum of 40 observations is required to determine the 2.5th and 97.5th percentiles, but the recommended minimum number of subjects for establishing a reference interval is 120.10 Suboptimal sample size is a limitation of the study reported here and also of most of the reference intervals published in the veterinary literature. Although extreme values were detected for analyte concentrations in a few plasma samples, those extremes were considered typical10 and were not excluded from the statistical analyses.

Plasma concentrations of only 3 analytes (total proteins, albumin, and creatinine) were normally distributed. The most frequent asymmetry with clinical biochemical data is positive skewness.10 For that condition, mathematically transformed values often fit a Gaussian distribution quite closely.10 In our study, this was true for plasma urea concentration and ALP activity after logarithmic transformation and for plasma glucose concentration after inverse transformation. However, simple mathematic transformation of the values of 7 other analytes did not yield a Gaussian distribution, and the corresponding reference intervals had to be defined by use of a nonparametric approach. This emphasizes the need to initially test the hypothesis of a Gaussian distribution for plasma biochemical results in a reference sample when establishing reference intervals.

Another objective of the study reported here was to assess the effect of age, sex, body weight, and housing condition on feline plasma analytes. No partitioning of data by age, body weight, or sex could be considered a priori because no information was available in the literature on the potential influence of these factors on analyte values. The effects of these factors were consequently determined in the study, but it was not possible to draw conclusions about proposed ageor body weight–related reference intervals. In the cats in our study, plasma glucose concentration increased with age, whereas plasma concentration of phosphates and albumin decreased with age. Similar results have been obtained for healthy humans during the aging process.28,29 Concentrations of plasma phosphates are also reportedly30,31 lower in mature Beagles than in young Beagles.

We also found an increase in plasma concentrations of albumin, creatinine, glucose, and total proteins with increasing body weight. A similar association between plasma creatinine concentrations and body weight has also been reported in children29 and dogs.32,f The influence of body condition on analyte values could not be evaluated in the study reported here because body fat content was not assessed. The higher body weight in the cats of our study is likely to be correlated with increased adiposity. Baseline concentrations of plasma glucose did not differ significantly between normal-weight and obese cats in another study.33 This discrepancy with our results could be explained by the difference in number of animals tested (94 in this study vs 15 in that other study33). However, our results are consistent with those described in other species.29,34 To our knowledge, an effect of body weight on plasma concentrations of albumin or total proteins has not been reported.29

In our reference sample, the plasma concentration of chloride was lower in indoor cats, whereas the activity of ALP and concentrations of CO2 and phosphates were higher. The cause of the difference is unknown and requires further study. Housing conditions can influence plasma biochemical results in dogs.35 Although significant differences were detected between mean values of analytes for indoor versus outdoor cats, these differences were considered to be clinically irrelevant. For example, the greatest difference in mean plasma ALP activity was 22%.

The results reported here provide systematically established and defined reference intervals for plasma analytes of cats in a clinical situation. However, it should be kept in mind that the findings cannot be extrapolated to other breeds of cats or applied to the results obtained from laboratory conditions other than those described here. The influence of factors affecting variation of plasma analytes in cats requires further study.

ABBREVIATIONS

DSH

Domestic shorthair

ALT

Alanine aminotransferase

ALP

Alkaline phosphatase

a.

Veterinary Clinic Quai d'Orleans, Tours, France.

b.

Lithium heparinate Microvette, 200 μL, Sarstedt, Nümbrecht, Germany.

c.

Vitros 250 chemistry system, Ortho-Clinical Diagnostics, Raritan, NJ.

d.

Performance Verifier I & II control sera, Ortho-Clinical Diagnostics, Issy-lès-Moulineaux, France.

e.

Systat, version 8.0, SPSS Inc, Chicago, Ill.

f.

Craig AJ, Seguela J, Queau Y, et al. Refining the reference interval for plasma creatinine in dogs: effect of age, gender, body weight and breed (abstr). J Vet Intern Med 2006;20:740.

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