J Vet Intern Med 2010;24:809–818

B r e e d De p e n d e n c y o f Re f e r e n c e I n t e r v a l s fo r P l a s m a B i o c h e m i c a l
V al ue s i n C at s
B.S. Reynolds, D. Concordet, C.A. Germain, T. Daste, K.G. Boudet, and H.P. Lefebvre

Background: Reference intervals (RI) are pivotal in clinical pathology. The influence of breed on RI has been poorly doc-
umented in cats.
Hypothesis/Objectives: RI for plasma biochemistry variables are breed-dependent in cats.
Animals: Five hundred and thirty-six clinically healthy, fasted, client-owned cats from 4 breeds: Holly Birman (n 5 132),
Chartreux (n 5 129), Maine Coon (n 5 139), and Persian (n 5 136).
Methods: Prospective observational study: Blood samples were collected from the cephalic vein into capillary tubes contain-
ing lithium heparin. Plasma glucose, urea, creatinine, total proteins, albumin, calcium, phosphate, sodium, potassium, chloride,
and total CO2 concentrations and the activities of alanine aminotransferase and alkaline phosphatase were assayed with a dry
slide biochemical analyzer. RI were defined as central 95% intervals bounded by the 2.5th and 97.5th percentiles. Data were
analyzed by a linear mixed effects model with type I error rate of 0.05.
Results: A significant (P o .05) breed effect was observed for 9/13 variables. The magnitude of the differences between
breeds could be clinically relevant for creatinine, glucose, and total protein. Age, body weight, sex, and housing conditions had
significant (P o .05) breed-related effects on different variables.
Conclusions and Clinical Importance: Breed-specific RI should be considered for cats.
Key words: Breeds; Clinical chemistry; Feline.

reed effects on biochemical variables have been re-

B ported for dogs.1–8 The dog provides an unique
model because there are 4300 breeds and the morpho-
Abbreviations:
ALP alkaline phosphatase
logical differences between breeds can be large with body
ALT alanine aminotransferase
weight (BW) varying from o1 kg (Chihuahua dog) to B Holly Birmans
460 kg (Irish Wolfhound). Although interindividual BUN blood urea nitrogen
phenotype differences are more limited in humans com- BW body weight
pared with those observed in dogs, there are racial C Chartreux
differences.9,10 In contrast, limited information is avail- CI confidence intervals
able for domestic cats. The number of breeds of cats is CLSI Clinical and Laboratory Standards Institute
also much more limited than in dogs: 55 feline breeds DSH domestic shorthair cats
are recognized for championship competition by the MC Maine Coons
International Cat Association (http://www.tica.org). P Persians
Moreover, about 60% of cat breeds are of recent lineage RI reference intervals
and were produced during the last 100 years.11 In addi-
tion, breed barriers are not so strictly defined in cats as in
dogs and outcrosses are permissible for some breeds of might be necessary at least for plasma creatinine which
very recent origin.11 Nevertheless, a breed effect on appears to be higher in the Holly Birman.15 Thirdly, re-
plasma biochemical values cannot be excluded for at cent data have demonstrated that genetic diversity in cats
least 3 reasons. Firstly, breed has been identified as a risk is broad and different clusters can be identified.16,17
factor for predisposition to specific clinical settings, such The primary objective of the prospective study re-
as Holly Birmans for feline infectious peritonitis12 or ported here was to assess the effect of breed on routine
Burmese for diabetes mellitus.13,14 Secondly, a breed- plasma analytes and their corresponding RI, in cats from
dependent stratification of reference intervals (RI) in cats 4 different breeds. A secondary objective was to assess
the effects of age, BW, sex, and housing conditions on the
From the Department of Clinical Sciences (Reynolds, Germain, biochemistry results.
Daste, Boudet, Lefebvre) and the Unite´ Mixte de Recherche 181
Physiopathologie et Toxicologie expérimentales, Institut National de
la Recherche Agronomique (Concordet, Germain, Lefebvre), Na- Materials and Methods
tional Veterinary School of Toulouse, Toulouse, France. Results were
This study was conducted between March 27 and November 28,
presented in part at the American College of Veterinary Internal
2007. Written consent was obtained from each owner of participat-
Medicine Congress, San Antonio, June 4–7, 2008. The study was per-
ing cats.
formed at the National Veterinary School of Toulouse, France.
Corresponding author: Herve P. Lefebvre, Department of Clinical
Sciences, National Veterinary School of Toulouse, 23 chemin des Cat Selection
Capelles, BP 87614, 31076 Toulouse cedex 03, France; e-mail:
h.lefebvre@envt.fr. Healthy purebred owned cats were prospectively recruited for
Submitted September 3, 2009; Revised March 8, 2010; this study. The participating owners/breeders were solicited by
Accepted April 16, 2010. phone contact. Heparinized blood samples were obtained from the
Copyright r 2010 by the American College of Veterinary Internal cats either in private homes, in a veterinary practicea or in 2 veter-
Medicine inary teaching hospitals.b,c Appointments were made with all
10.1111/j.1939-1676.2010.0541.x participating owners/breeders, who were instructed to withhold
810 Reynolds et al

food from their cats 12–24 hours before the scheduled blood collec-
tion. Breed, age, sex, fasted status, housing conditions, and medical
history were recorded for each cat. It was then weighed and a phys-
ical examination performed just before blood collection. Two to 3
months later, the owners/breeders of participating cats were con-
tacted by phone and asked about the apparent health status of their
animal. Cats were not included in the study if they were o6 months
old, in a nonfasted state, not purebred, had a history of any disease,
had received any drug treatment during the 4 weeks before sample
collection, if they showed clinical signs of illness on the day of blood
collection, or were not reported to be healthy at phone follow-up.
Sampling Procedure
Assays
All plasma biochemical assays were performed with a dry-slide
technology analyzere (Table 1) in the clinical pathology laboratory
of the National Veterinary School of Toulouse. The plasma analytes
tested were glucose, urea, creatinine, total proteins, albumin, cal-
cium, phosphate, sodium, potassium, chloride, and total CO2,
together with the activities of alanine aminotransferase (ALT) and
alkaline phosphatase (ALP). A detailed description of the valida-
tion of these assays in the authors’ laboratory, under the same
conditions, has been published previously (Table 1).19 Laboratory
values are reported in SI units, as recommended by the Clinical and
Laboratory Standards Institute (CLSI). Conversion factors for US
units are provided in Appendix 1.

All blood samples were collected by the same investigator. Blood

was obtained from a cephalic vein with a 25-G needle and two to
three 200 mL capillary tubes that contained heparinate lithium (lith-
ium heparinate Microvette, 200 mL),d as described elsewhere.18,19
Sample Processing
Blood samples were centrifuged (3,000  g for 10 minutes) within
30 minutes after collection. The plasma supernatant was then im-
mediately harvested and stored at 201C until assayed within 5 days
in the same laboratory. Sample stability in such conditions has been
described previously.19 On the days of testing, the plasma samples
were allowed to thaw at room temperature for 30 minutes before
being assayed. Sample quality was recorded after centrifugation at
the time of collection and again at the time of the assay.
Statistical Analysis
Identification of outliers and determination of RI were per-
formed according to the current CLSI guidelines.20 Native and
Box-Cox transformed data in each breed were first tested for nor-
mality by use of the Anderson-Darling test. The data set was then
examined for outliers in each breed separately. Tukey’s criterion
was applied when the distribution was Gaussian. When the data
distribution remained non-Gaussian after Box-Cox transformation,
this concept was not applicable and visual inspection of values in
both tails of the distribution was used. When a value was deemed
questionable, the following criteria were used to decide to remove a
cat from the study: any abnormality concerning medical history or
clinical examination, an atypical value for more than one analyte

Table 1. Test characteristics for analytes,a repeatability, and within-laboratory imprecision estimates for control
solutions of plasma analytesb measured by use of the analyzer.
Repeatability Imprecision
Analytic Control
Analyte (unit) Methodology Range Concentrations SD CV (%) SD CV (%)
Glucose (mmol/L) Colorimetric (glucose oxidase) 1.1–34.7 4.8 0.03 0.7 0.17 3.8
15.9 0.07 0.5 0.23 1.4
Urea (mmol/L) Colorimetric (urease) 0.7–42.8 5.6 0.09 1.4 0.08 1.2
16.4 0.19 0.9 0.14 0.7
Creatinine (ı́mol/L) Enzymatic (creatinine amidinohydrolase) 4–1,238 87 0.9 0.9 3.0 3.1
523 3.5 0.7 8.1 1.6
Total proteins (g/L) Colorimetric (cupric tartrate) 20–110 43 1.2 2.8 0.6 1.5
68 1.9 2.7 1.0 1.5
Albumin (g/L) Colorimetric (Bromocresol green) 10–60 25 0.4 1.5 0.6 2.5
43 0.5 1.1 0.7 1.6
Calcium (mmol/L) Colorimetric (Arsenazo III) 0.25–3.49 2.30 0.021 0.9 0.032 1.3
3.00 0.014 0.4 0.033 1.0
Phosphates (mmol/L) Colorimetric (molybdate) 0.16–4.20 1.06 0.012 1.1 0.017 1.6
2.41 0.010 0.5 0.015 0.6
Sodium (mmol/L) Potentiometric (ion selective electrode) 75–250 124 0.7 0.6 2.7 2.3
147 0.8 0.5 2.1 1.5
Chloride (mmol/L) Potentiometric (ion selective electrode) 50–175 86 0.5 0.7 1.5 1.8
118 0.6 0.6 2.7 2.5
Potassium (mmol/L) Potentiometric (ion selective electrode) 1–14 3 0.0 0.7 0.0 1.4
5.6 0.0 0.8 0.1 2.0
Total CO2 (mmol/L) Enzymatic (phosphoenolpyruvate carboxylase) 5–40 13 0.6 2.5 0.7 3.0
27 0.7 5.3 0.7 4.8
ALT (U/L) Enzymatic (L-alanine and a-acetoglutarate substrate) 3–1,000 37 0.9 2.1 1.6 3.7
204 1.4 0.8 3.3 1.8
ALP (U/L) Enzymatic (p-nitrophenyl phosphate substrate) 20–1,500 74 1.6 1.5 1.8 1.8
469 5.8 1.0 13.6 2.5

ALT, alanine aminotransferase; ALP, alkaline phosphatase.

a
From Instructions for use, Vitros chemistry products, Ortho-Clinical Diagnostics Inc.
b
From Reynolds et al.19
 Reference Intervals for Cats 811

from the same cat and distance of an extreme value from the closest Twenty-five batches, corresponding to the whole popu-
value observed. lation of tested cats, were analyzed.
RI were defined as central 95% intervals bounded by the 2.5th
and 97.5th percentiles. Upper and lower limits of RI with their 90%
confidence intervals (CI) were determined in the global population
and in each breed by a nonparametric approach. The potential rel-
evance of a breed specific RI was further addressed. Lower and Identification of Outliers, Distribution of Plasma
upper limits of the RI with corresponding 90% CI for each breed Concentrations, and Determination of RI in
and the overall population were first graphically represented for Each Breed
visual assessment of between-breed discrepancies. For plasma
creatinine, which appeared to be the most relevant variable in terms
No abnormality concerning medical history or clinical
of breed differences, appropriate partitioning criteria were applied examination could be found to warrant discarding any
to each pair of breeds to assess the need for separate RI. Partition- cat identified with possible outlying values in any of the
ing, by comparing the breeds 2 by 2, was indeed considered breeds. Eleven cats were, however, excluded from the
necessary if any of the 4 proportions (2 at the lower and 2 at the data set based on the other criteria: 4 (1 B, 3 C) with
upper end of the distributions) outside the common reference limits atypical values for 2 analytes, 7 (3 C, 2 MC, and 2 P) with
were 4.1% or 0.9%.21 an extreme value distant from the closest observed val-
Effects of breed and other covariables on plasma variables were ues. Consequently, 525 cats were used for further data
tested using a statistical software package.22 A value of P o .05 was analyses. Detailed descriptive statistics of the population
considered significant. Age and BW in each breed were graphically
for each of the 4 breeds are given in Table 2. Of the 52 (13
assessed by boxplots and compared by ANOVA. The effects of
breed, age, BW, sex, and housing conditions on plasma variables
variables  4 breeds) breed-specific distributions of
were tested by the following linear mixed effects model in which the plasma variables tested for normality, 14 were Gaussian.
owner appears as a random effects factor: Box-Cox transformation yielded a Gaussian distribution
for 20 other variables. The remaining 18 could not be
Yijklm ¼m þ Owneri þ Breedj þ a Ageijklm þ Sexk transformed to fit a Gaussian distribution (Table 3).
þ b BWijklm þ Housingl þ aj Age Corresponding lower and upper limits of the RI for
þ ðBreed  SexÞjk þ bj BW
tested plasma variables with 90% CI are provided for
each breed in Table 3. They are graphically represented
þ ðBreed  HousingÞjl þ eijklm in Figure 1 as well as those corresponding to the overall
population for plasma glucose, creatinine, and total pro-
where Yijklm is the value expressed in SI units of plasma variable Y teins. Results of the application of objective partitioning
for Cat m, with Owner i, Breed j, Sex k, and Housing l; m is a con- criteria to each pair of breeds to assess the need for sep-
stant term; Owneri is a random effect factor assumed to be N (0;
arate RI according to breed for plasma creatinine are
s2owner); Age and BW are continuous covariables, a and b are the
slope coefficients for Age and BW irrespectively of the breed; Sexk is provided in Table 4.
the differential effect of level k for the Sex factor and Housingl is the
differential effect of level l for the Housing factor; aj (Breed  Sex)jk, Table 2. Descriptive statistics of the reference sample
bj, BW, and (Breed  Housing conditions)jl denote an interaction group in each breed.
between Breed and Age, Breed and Sex, Breed and BW, and Breed
and Housing, respectively; a 1 aj and b 1 bj are the slope coeffi- Results
cients for Age and BW in Breed j, respectively; eijklm is the
Variable B C MC P
residual term of the model assumed to be N (0; s2).
When a significant interaction between a covariable and breed Number of cats 131 123 137 134
was evidenced, the effects of age, BW, sex, or housing on plasma Number of owners/ 18 46 17 25
variables were assessed breed by breed. Otherwise these covariables breeders
effects were assessed on the global sample. Sex (%)
Posthoc comparison tests were not performed because all com- F 93 (71) 76 (62) 97 (71) 94 (70)
parisons were a priori planned. Therefore, no correction for M 38 (29) 47 (38) 40 (29) 40 (30)
multiple comparisons was needed to control the overall type I er- NF 17 (13) 18 (15) 19 (14) 17 (13)
ror. All plasma variables moreover were analyzed separately as a NM 24 (18) 21 (17) 12 (9) 11 (8)
single plasma analyte can be assayed and interpreted to confirm or Housing conditions (%)
infirm a clinical hypothesis. I 55 (42) 36 (29) 49 (36) 91 (68)
O 76 (58) 87 (71) 88 (64) 43 (32)
Age (years)
Results mSD 4.03.0 3.82.9 3.12.2 3.92.8
Median 3.4 2.9 2.4 3.4
Reference Sample Group and Assays
Minmax 0.5–15.2 0.6–15.6 0.5–11.8 0.5–12.7
Five hundred and seventy-one purebred cats were BW (kg)
sampled. Five hundred and thirty-six of these (Holly mSD 3.40.9 4.91.4 4.81.4 3.20.8
Birmans [B]: n 5 132; Chartreux [C]: n 5 129; Maine Median 3.3 4.6 4.5 3.2
Minmax 1.8–6.0 2.4–9.5 2.1–9.4 1.1–5.7
Coons [MC]: n 5 139, and Persians [P]: n 5 136) met the
inclusion criteria. They belonged to 97 different owners/ B, Holly Birman; C, Chartreux; MC, Maine Coon; P, Persian; F,
breeders. Hemolysis, lipemia, or clot was not observed in total number of females; M, total number of males; NF, number of
any plasma sample. One plasma sample, belonging neutered females; NM, number of neutered males; I, strictly indoor;
to a cat that was subsequently excluded, was icteric. O, some access outside.
 812

Table 3. Reference intervals for plasma analytes in healthy Holly Birman (B), Chartreux (C), Maine Coon (MC), and Persian (P) cats.
B C MC P

LL (90% CI) LL (90% CI) LL (90% CI) LL (90% CI)

Analyte Distribution UL (90% CI) Distribution UL (90% CI) Distribution UL (90% CI) Distribution UL (90% CI)
Glucose (mmol/L) NG 3.4 (3.0–4.0) BCG 4.2 (4.1–4.3) NG 3.4 (3.2–3.8) BCG 3.5 (2.9–3.8)
6.1 (5.8–7.0) 9.3 (8.9–10.5) 9.1 (7.7–11.9) 6.8 (6.1–7.1)
Urea (mmol/L) G 7.2 (6.7–7.6) BCG 4.5 (4.0–5.0) G 6.1 (5.9–6.4) BCG 4.7 (4.3–5.1)
12.8 (12.5–14.9) 10.0 (9.8–13.8) 11.2 (10.7–12.0) 9.4 (9.2–11.4)
Creatinine (mmol/L) BCG 91 (88–98) G 80 (61–88) G 77 (66–83) BCG 80 (66–87)
239 (230–255) 189 (178–212) 192 (181–216) 164 (159–208)
Proteins (g/L) BCG 64 (56–65) G 64 (62–65) G 59 (55–60) G 59 (55–62)
103 (101–109) 88 (86–90) 85 (82–91) 83 (82–88)
Albumin (g/L) G 26 (23–28) G 25 (23–26) G 23 (21–24) G 24 (21–25)
37 (37–42) 38 (37–39) 36 (35–40) 39 (38–40)
Calcium (mmol/L) G 2.35 (2.32–2.42) G 2.39 (2.36–2.41) G 2.26 (2.16–2.32) BCG 2.31 (2.14–2.39)
2.84 (2.78–2.98) 2.79 (2.77–2.85) 2.86 (2.75–3.15) 2.93 (2.91–3.47)
Phosphate (mmol/L) BCG 1.08 (0.98–1.17) BCG 1.05 (0.89–1.22) BCG 0.90 (0.79–1.08) BCG 1.13 (1.06–1.22)
2.51 (2.29–3.07) 2.74 (2.41–3.04) 2.79 (2.56–3.19) 2.51 (2.38–2.97)
Reynolds et al

Sodium (mmol/L) NG 151 (149–152) NG 152 (151––152) NG 151 (151–152) NG 151 (148–152)
163 (160–166) 162 (161–164) 161 (159–163) 164 (163–167)
Potassium (mmol/L) NG 3.4 (3.1–3.5) NG 3.2 (3.2–3.5) NG 3.4 (3.1–3.5) NG 3.4 (3.4–3.5)
4.8 (4.7–5.2) 4.6 (4.5–4.8) 4.5 (4.3–4.8) 4.9 (4.4–5.3)
Chloride (mmol/L) NG 114 (112–116) NG 117 (116–118) NG 119 (117–119) NG 117 (116–118)
129 (127–135) 129 (127–130) 129 (128–132) 128 (127–133)
CO2 (mmol/L) NG 14 (12–14) NG 15 (14–16) NG 14 (11–15) NG 15 (14–16)
22 (21–23) 24 (23–24) 22 (22–23) 23 (23–25)
ALT (U/L) BCG 27 (21–35) BCG 25 (15–30) BCG 24 (16–26) BCG 25 (18–28)
148 (131–185) 157 (127–209) 103 (87–158) 147 (114–214)
ALP (U/L) BCG 29 (26–32) BCG 29 (26–32) BCG 28 (26–31) BCG 26 (20–29)
93 (79–123) 93 (82–133) 107 (102–127) 105 (88–120)

G, Gaussian; BCG, Gaussian after Box-Cox transformation; NG, nonGaussian; LL, Lower limit; UL, upper limit; 90% CI, 90% confidence intervals; ALT, alanine aminotransferase; ALP, alkaline
phosphatase.
 Reference Intervals for Cats 813

A Breed Effect
Significant differences between breeds were evident for
BW (P o .001) but not for age (P 5 .08) (Fig 2). Some
interactions between covariables and breed were statisti-
cally significant for some analytes (Table 5). A breed
effect was demonstrated for plasma glucose (P o .001),
urea (P o .001), creatinine (P o .001), total protein (P
o .001), albumin (P o .001), calcium (P 5 .007), potas-
2 3 4 5 6 7 8 9 10 11 12 sium (P 5 .04), total CO2 (P o .001), and activity of
Glucose (m m ol / L) ALT (P 5 .006) but not for plasma sodium (P 5 .052),
chloride (P 5 .074), phosphate (P 5 .435), and ALP ac-
B tivity (P 5 .243) (Tables 6–8).

Effects of Age, BW, Sex, and Housing Conditions on

Plasma Variables
Results are provided in Tables 6–8.

50 100 150 200 250 Discussion

Creatinine (µm ol / L) This study demonstrated a breed effect for 9/13 plasma
variables tested. Comparison of rationally established RI
C further revealed discrepancies between breeds that could
be of clinical relevance for some plasma analytes.
A major advantage is that the large number of animals
included in this study allowed RI to be calculated ac-
cording to the current CLSI guidelines.20 Moreover, for
most of the published RI for cats, limited information is
available regarding the reference sample group and the
50 60 70 80 90 100 110 methods of calculation. As also stated in the CLSI guide-
Total proteins (g / L) lines, all the preanalytical and analytical steps (fasting,
blood collection technique, specimen processing, and as-
Figure 1. Lower and upper limits with 90% confidence intervals of
says) were here standardized and controlled. However, it
reference intervals for the global population (white squares), Bir-
mans (black triangles), Chartreux (white circles), Maine-Coons should be kept in mind that our results cannot be ex-
(black squares), and Persians (white triangles) for plasma glucose trapolated to laboratory conditions other than those
(A), creatinine (B), and total proteins (C). Dotted vertical lines rep- described.
resent the value for lower and upper limits of the reference interval This study also draws attention to potential issues re-
for the global population. garding RI establishment, especially the selection of

Table 4. Partitioning of RI for plasma creatinine according to breed.

Proportions (%) Outside the Common Reference Limits
Common RI
Combined Limits (mmol/L) B C MC P Decision
B1C Lower 5 87 0 4.9 NA NA Partition
Upper 5 229 5.3 0 NA NA
B1MC Lower 5 80 0 NA 4.4 NA Partition
Upper 5 228 5.3 NA 0 NA
B1P Lower 5 84 0 NA NA 3 Partition
Upper 5 228 5.3 NA NA 0
C1MC Lower 5 78 NA 1.6 2.9 NA Combined
Upper 5 192 NA 2.4 2.2 NA
C1P Lower 5 81 NA 2.4 NA 2.2 Partition
Upper 5 181 NA 4.9 NA 0.7
MC1P Lower 5 78 NA NA 2.9 1.5 Partition
Upper 5 186 NA NA 4.4 0.7

A common RI is established for each pair of breed. Proportions of cats in each breed at the lower and upper end of the distributions outside
the limits of the common RI are determined. Separate RI have to be considered when 1 of the 4 proportions observed is either 4.1 or 0.9%;
Combined RI is appropriate if all the proportions fall between 1.8 and 3.2%.21
B, Holly Birman; C, Chartreux; MC, Maine Coon; NA, not applicable; P, Persian; RI, reference interval.
814 Reynolds et al

A 10 Table 5. P value for the statistically significant interac-

tions in the linear mixed effects model used for testing the
9 plasma variables.

8 Interaction of the Breed Effect with the Following

Covariable Effect
7 Tested Variables Age Sex Body Weight Housing
Glucose .26 .96 .34 .005
BW (kg)

6
Urea .73 .56 .50 .043
Creatinine .11 .54 .050 .016
5 Total proteins .55 .18 .50 .70
Albumin .29 1.00 .024 .29
4 Calcium .40 .48 .69 .11
Phosphates .055 .98 .45 .010
3 Sodium .21 .90 .60 .017
Potassium .58 .46 .98 .013
2 Chloride .26 .33 .93 .42
Total CO2 .55 .25 .43 .24
1 ALT .009 .31 .66 .86
B C MC P ALP .055 .72 .16 .72
BREED
A significant interaction between a covariable and breed implies
B 20 that the effects of this covariable on the plasma analyte tested
changes is different between breeds. P values for statistically signifi-
cant interactions appear in bold.
ALT, alanine aminotransferase; ALP, alkaline phosphatase.

15
fold) and BW (from 3.5- to 5.2-fold). It is worth noting
that no difference in age between the 4 breeds was evi-
denced. By contrast, the observed differences in BW
AGE (y)

between some breeds were expected and unavoidable as

10
5 Selection of reference individuals is also a complex is-
0 tions and were considered healthy by their owners/
B C MC P
BREED
Figure 2. Box-plots illustrating body-weight in kilograms (A) and
age in years (B) for each of the 4 breeds. B, Holly Birman; C, Chart-
reux; MC, Maine Coon; P, Persian.
reference individuals, treatment of outlying observa-
tions, and the partitioning criteria.
B, C, MC, and P were selected as reference sample
groups in this study for 2 main reasons. Firstly, recent
studies have demonstrated that, in accordance with their
phenotypic differences, these breeds are also clearly dis-
tinct on a genetic basis.17 Secondly, the use of common
breeds of cats was necessary to obtain sample groups of
appropriate size and, in particular, to reach the minimum
number of 120 subjects by breed recommended for
establishing RI.23 In addition, the choice of well-
represented feline breeds was deemed relevant with re-
gard to potential application of the results in clinical
settings. Another advantage was that each tested breed
population showed a wide range of age (from 24- to 30-
they were intrinsic breed-dependent characteristics. Nev-
ertheless, these differences in BW do not explain the
breed-related variations, as both factors were taken into
account in the statistical model.
sue as the definition of health is not straightforward. As
recommended by the guidelines of the CLSI, the subjects
here were not ill and did not require hospitalization or
treatment.24 They underwent yearly veterinary examina-
breeders. Any history of clinical signs of illness or
medication administration was defined as noninclusion
criteria and only cats without any clinical sign were in-
cluded. Moreover, all owners/breeders were contacted by
phone 2–3 months after the sampling day to check that
all the cats had remained healthy during the postsam-
pling period. Finally, it can be helpful to identify any
outlying values, as was done here. Eleven of the 536
(2.1%) included cats were considered to show aberrant
values and were removed from the data set.
The possibility of establishing separate RI, ie, parti-
tioning, should also be considered before the process of
analyzing the data.20 When only 2 subclasses (eg, male
and female) are compared, 2 methods (the Harris/Boyd
approach for Gaussian distributions and an alternative
approach for non-Gaussian ones) are used.20 However,
no method has been proposed when more than 2
subclasses (here 4 breeds) need to be compared. Although
not ideal, we therefore decided to use a partitioning crite-
ria, as proposed previously, on each pair of breeds.21 This
partitioning was only performed for plasma creatinine,
 Reference Intervals for Cats 815

Table 6. Slope coefficients for the continuous variables (age and body weight) and differential effects for the categor-
ical variables (breed, sex, housing conditions) of the linear mixed effect model used for the statistical analysis of the
plasma glucose, urea, creatinine, total proteins, and albumin with corresponding P values.
Tested Variable m Breed Age Sex Body Weight Housing
Glucose (mmol/L) 4.59 B: 0.32 P 5 .20 F: 0.08 10.17 P 5 .088
C: 0.02 M: 10.08 P 5 .002
MC: 10.54 P o .001
P: 0.20
P o .001
Urea (mmol/L) 7.39 B: 11.72 10.01 F: 0.10 P 5 .053 P 5 .16
C: 0.81 P o .001 M: 10.10
MC:0.48 P 5 .009
P: 0.44
P o .001
Creatinine (mmol/L) 87.5 B: 4.25 11.86 F: 12.01 B: 120.24 P 5 .66
C: 4.74 P o .001 M: 2.01 C: 18.45
MC:6.38 P o .001 MC: 18.07
P: 115.37 P: 13.82
P o .001 P o .001
Total proteins (g/L) 70.2 B: 15.76 10.49 P 5 .90 P 5 .099 P 5 .51
C: 0.92 P o .001
MC: 1.43
P: 3.41
P o .001
Albumin (g/L) 27.9 B: 0.52  0.25 F: 10.36 B: 11.69 P 5 .27
C: 0.06 P 5 .006 M: 0.36 C: 11.14
MC: 11.10 P 5 .002 MC: 10.43
P: 0.52 P: 11.52
P o .001 P o .001

The slope coefficients and differential effects are only given when the effect is statistically significant.
For example, the general regression equation for plasma creatinine in male Birman cats will be: Plasma creatinine (mmol/L) 5 87.5  4.25 1
1.86  Age (years)  2.01 1 20.24  BW (kg) 5 81.24 1 1.86  Age (years) 1 20.24  BW (kg).
For a male Birman cat (10.0 years, 4.0 kg), the estimated plasma creatinine concentration will be: Plasma creatinine (mmol/L) 5 81.24 1
1.86  10 1 20.24  4 5 181 mmol/L (or 2.0 mg/dL).
m, overall mean for the global population; B, Holly Birman; C, Chartreux; MC, Maine Coon; P, Persian; F, female; M, male.

Table 7. Slope coefficients for the continuous variables (age and body weight) and differential effects for the categor-
ical variables (breed, sex, housing conditions) of the linear mixed effect model used for the statistical analysis of the
plasma calcium, phosphate, sodium, potassium, chloride, and total CO2, with corresponding P values.
Tested Variable m Breed Age Sex Body Weight Housing
Calcium (mmol/L) 2.60 B: 0.027 0.013 P 5 .12 P 5 .27 P 5 .32
C: 10.003 P o .001
MC: 0.015
P: 10.040
P 5 .007
Phosphates (mmol/L) 2.17 P 5 .44 0.062 F: 0.11 0.057 B I: 10.12
P o .001 M: 10.11 P o .001 O: 0.12
P o .001 C I: 0.05
O: 10.05
MC I: 10.06
O: 0.06
P I: 10.04
O: 0.04
P 5 .019
Sodium (mmol/L) 155.2 P 5 .052 P 5 .51 P 5 .94 P 5 .13 P 5 .71
Potassium (mmol/L) 3.87 B: 10.09 P 5 .15 P 5 .21 P 5 .44 B I: 10.14
C: 0.06 O: 0.14
MC: 0.12 C I: 10.03
P: 10.10 O: 0.03
P 5 .040 MC I: 0.02
O: 10.02
P I: 10.02
O: 0.02
P 5 .001
816 Reynolds et al

Table 7. (Continued).

Tested Variable m Breed Age Sex Body Weight Housing

Chloride (mmol/L) 123.5 P 5 .074 10.13 F: 10.30 P 5 .075 P 5 .48
P 5 .022 M: 0.30
P o .001
Total CO2 (mmol/L) 18.2 B: 0.83 P 5 .078 F: 0.30 10.13 I: 10.31
C: 0.31 M: 10.30 P 5 .042 O 5  0.31
MC: 1.04 P o .001 P 5 .017
P: 3.41
P o .001

The slope coefficients and differential effects are only given when the effect is statistically significant.
For example, for a male Maine Coon cat (9.0 years, 8.0 kg) living strictly indoors, the estimated plasma phosphate concentration will be:
Plasma phosphate (mmol/L) 5 2.17  0.062  9 1 0.11  0.057  8 1 0.06 5 1.33 mmol/L.
m, overall mean for the global population; B, Holly Birman; C, Chartreux; MC, Maine Coon; P, Persian; F, female; M, male; I, strictly
indoor; O, some access outside.

Table 8. Slope coefficients for the continuous variables (age and body weight) and differential effects for the categor-
ical variables (breed, sex, housing conditions) of the linear mixed effect model used for the statistical analysis of the
plasma alanine aminotransferase (ALT) and alkaline phosphatase (ALP) with corresponding P values.
Tested Variable m Breed Age Sex Body Weight Housing
ALT (U/L) 77.7 B: 117.1 B: 11.05 F: 11.41 4.15 P 5 .27
C: 10.03 C: 0.63 M: 1.41 P o .001
MC: 13.8 MC: 10.00 P 5 .002
P: 3.33 P: 3.47
P 5 .006 P 5 .006
ALP (U/L) 78.6 P 5 .24 1.56 F: 5.94 4.85 P 5 .17
P o .001 M: 15.94 P o .001
P 5 .004

The slope coefficients and differential effects are only given when the effect is statistically significant.
For example, for a female Persian cat (3.0 years old, 3.5 kg), the estimated plasma ALT activity will be: Plasma ALT (U/L) 5 77.7  3.33 
3.47  3 1 1.41  4.15  3.5 5 51 U/L.
m, overall mean for the global population; B, Holly Birman; C, Chartreux; MC, Maine Coon; P, Persian; F, female; M, male.

which was the variable that appeared to be the most
relevant to propose breed-specific RI. A need for parti-
tioning was clearly demonstrated for all the 2 by 2 breed
combinations except between MC and C.
Moreover, differences assessed by visual inspection
(Fig 1) of 90% CI for upper limits of breed-specific RI
appear large enough to be considered clinically relevant
for 3 plasma variables, namely creatinine, total proteins,
and glucose, in pure-bred cats. This could be especially
relevant when plasma creatinine is used as a first-line
diagnostic test for screening early chronic kidney disease.
The same issue exists in canine species where plasma
creatinine concentration is known to be higher in Grey-
hounds than in other dogs.8 Similar differences have also
been observed in humans where plasma creatinine con-
centration has been reported to be higher in non-
Hispanic black Americans, lower in non-Hispanic whites
and lowest in Mexican Americans.10 Race-related differ-
ences in serum proteins have also been described in
humans.25 By contrast, the albumin RI were similar from
one breed to another.
A possible explanation for the observed differences in
plasma glucose would be a stress-related increase, as
described previously in felines.26 The level of stress is
quite unpredictable and difficult to assess by observa-
tion.27 However, it was likely to be the same in all the
breeds tested here because the sampling procedure was
strictly identical. Moreover, to our knowledge, C and
MC have not been reported to be more susceptible to
stress than B and P. Higher fasting plasma glucose con-
centrations were reported in human ethnic groups that
also showed a higher susceptibility to diabetes melli-
tus9,28 and a predisposition of Burmese cats to diabetes
mellitus has also been described.13,14
It could be of interest to compare the RI established in
the present study with those available in the veterinary
literature. The only original publication regarding RI for
the same variables is available for domestic shorthair cats
(DSH) cats.19 The upper limit of the RI for plasma
creatinine, glucose, and total proteins was 207 mmol/L
(2.3 mg/dL), 8.2 mmol/L (148 mg/dL), and 85 g/L. For
example, if the DSH upper limit for plasma creatinine
had been applied, 13/131 (10%) of B tested in the present
study would have been considered azotemic. Such a com-
parison, however, should be performed with caution
because of the lower number (n 5 95) of animals and
the different methodology for RI calculation.
A linear mixed effects model was used here to assess
the effect of covariables on the tested plasma analytes.
The owner was considered as a random effects factor.
The owner effect should indeed be treated as a random
effects factor as a sample of owners has been drawn
 Reference Intervals for Cats
within a population of owners. It is obvious that the
owner may impact the environmental conditions of cats
and that response variables for cats belonging to 1 owner
could be more related than those of cats from different
owners. As the breed effect is adjusted on the owner
effect, the difference of number of owners between breeds
may not lead to bias. Indeed, the statistical results dem-
onstrated that the breed effect was evidenced for 9/13
variables when the owner effect was taken into account.
In other words, the breed effect was not confounded with
the owner effect.
Another objective of this study was to assess the effects
of other covariables (ie, age, BW, sex, and housing con-
ditions) on plasma values. Interestingly, the linear mixed
effects model also showed statistically significant interac-
tions between the breed effect, and the effects of age, BW,
and housing conditions, but not sex. A statistically sig-
nificant interaction between the breed and a covariable
means that the effect of this covariable on the analyte
tested is dependent on the feline breed. For example, for
plasma creatinine concentration, when all the other co-
variables are unchanged, a difference of 2 kg of BW
between 2 individuals will lead to a difference in the basal
plasma creatinine concentration of about 40 (0.45) and 8
(0.09) mmol/L (mg/dL) in B and P, respectively.
A statistically significant effect of age, sex, BW, and
housing conditions was observed for 9/13, 9/13, 7/13,
and 3/13 plasma variables, respectively. However, when
the slope coefficients and the differential effects were in-
spected, it could be seen that these effects would be
clinically irrelevant except for the effect of BW on plasma
creatinine in B.
In conclusion, breed-dependent RI should be consid-
ered in feline clinical pathology, at least for some of the
plasma analytes tested, such as creatinine, total proteins,
and glucose. Age, sex, and housing conditions appear to
be minor factors of variations of plasma variables in the 4
breeds tested. Conversely, BW was shown to affect
plasma creatinine, especially in B. Further investigations
are needed to understand the mechanisms underlying
these differences.
Footnotes
a Near Eastern origin of cat domestication. Science 2007;27:519–523.
Veterinary Clinic Quai d’Orleans, Tours, France
b 17. Lipinski MJ, Froenicke L, Baysac KC, et al. The ascent of
National Veterinary School of Toulouse, Toulouse, France
c cat breeds: Genetic evaluations of breed and worldwide random-
National Veterinary School of Nantes, Nantes, France
d breed populations. Genomics 2008;91:12–21.
Sarstedt, Nümbrecht, Germany
e 18. Reynolds BS, Boudet KG, Faucher MR, et al. Comparison
Vitros 250 chemistry system, Ortho-Clinical Diagnostics, Raritan,
NJ
Acknowledgments
The study was supported in part by Royal Canin SAS,
Centre de Recherches, Aimargues, France.
The authors thank Mrs C. Moncelon and A. Dutech,
Drs P. Brillard, A. Gambier, and C. Arpaillange for tech-
nical assistance.
817
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