Establishment of reference intervals of haematology and biochemistry analytes in ICR mice of different ages

Abstract

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Outbred stocks of mice are widely used in pre-clinical research as these animals possess a diversified genetic background when compared with inbred strains of mice. It is crucial to assess particular alterations in the physiological and functional profiles of laboratory animals using haematological and biochemical indicators. These values can also differ between laboratories because they are influenced by many different factors. We aimed to provide normal values and reference intervals for selected haematology and biochemistry analytes of 570 ICR mice at three different ages: 6–8 weeks, 10–14 weeks and 6–9 months. Reference values were calculated by non-parametric methods. For comparisons between sexes, the independent-sample t-test and Mann–Whitney test were employed, and analysis of variance was used for age differences. The findings of the study revealed age-related declines in haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin concentrations. Mice aged 6–9 months had statistically higher platelet counts in their blood than mice of other ages. The white blood cell count had a significant age effect and progressively decreased with age. As mice get older, the percentage of neutrophils, monocytes and basophils increases, but the percentage of lymphocytes decreases. For the biochemical values, age-related significant differences in glucose, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and albumin concentrations were found. It was also found that creatinine concentrations were comparable across all age ranges. The values presented in the present work can be used as a reference to interpret clinical pathology data for other studies and to evaluate health status.

Keywords

Haematology biochemistry reference interval ICR mice age sex

Introduction

Laboratory mice are widely used in biomedical research, since they profoundly contribute to our understanding of biological phenomena. As a result, these mice are useful in the establishment of new diagnostic and therapeutic strategies.^1,2 Outbred stocks of mice possess a diversified genetic background when compared with inbred strains of mice.^3
–5 An outbred stock is defined as a restricted colony of animals with a limited increase in the inbreeding coefficient of <1% per generation and a high degree of genetic variability.^6,7 This intended diversity enables the mouse phenotype to closely model the heterogeneity encountered in the intended general human population.

The ICR mouse is an albino mouse strain that originated from animals at the Centre Anticancereus Romand in Switzerland. Later, since this strain was distributed from the Institute of Cancer Research in the USA, it was named ICR after the initial letters of the institute. Several lineages of ICR mice were developed worldwide for research purposes^4,5,8 ICR is a highly reproductive strain of experimental mice and is widely used in pharmacology, toxicology and stem cell studies.⁹ Differences in the phenotypes of ICR mice have arisen between newly established stocks (founder effects) in different places worldwide and those that have been bred for a long time (drift effects). Zydus Research Centre, India has established ICR stock using Charles River Laboratories breeders, and replacing breeders at regular interval. This mouse stock is mainly used for in-house toxicology, pharmacology and immunology research.

Toxicology studies require the collection and interpretation of haematology and clinical chemistry analytes at various time intervals during the course of a study.¹⁰ To interpret the data from such experiments, it is vital to know the normal ranges for the haematological and biochemical analytes of these mice since underlying pathologies can influence metabolism and alter the results obtained in experimental procedures. Limited haematology and clinical biochemistry information is available for ICR mice through a few studies.^11
–13 These values can be affected by many factors, such as age, sex, nutrition, animal housing, circadian rhythm, daily activity, stress, sexual cycle, et cetera. This study’s goal is to give researchers normal values and age-related reference intervals for several haematological and biochemical analytes of ICR mice bred under local standards at Zydus Research Centre. Over a number of years, we collected and developed an age-specific data set of haematology and biochemistry analytes for both sexes from the routine health monitoring of breeding colonies. The information used here was gathered from ICR mice that were 6–8 weeks, 10–14 weeks and 6–9 months old. Nearly all of the significant alterations in physiological development that occur during life are covered by these data. We have generated reference intervals for several measurands for three different age ranges, which can be used as reference data set for interpretation in non-clinical studies.

Materials and methods

Animals and diets

The ICR mice used for the data collection were bred and issued from Animal Research Facility of Zydus Research Centre in Ahmedabad, India. Animals were maintained in individually ventilated cages (IVCs) (ventilation rate set at 40–50 air changes per hour) with a 12/12 h light/dark cycle, with a controlled room temperature of 23°C ± 2°C and relative humidity conditions of 50% ± 20%. Except when otherwise noted, the mice received unlimited access to a standard chow diet (2018 Teklad global 18% protein rodent diets, Inotiv) and reverse osmosis-treated water. All health monitoring procedures met Committee for the Control and Supervision of Experiments on Animals requirements and received Institutional Animal Ethics Committee approval. In this investigation, the data collected from 570 ICR mice (190 mice/age range) at ages 6–8 weeks, 10–14 weeks and greater than 6 months are included.

Selection of animals

As part of the breeding colony’s health monitoring programme, a random sample of mice had their haematology and biochemistry analytes tested. In each health monitoring study, 60 mice (30 male and 30 female) were randomly chosen from three age ranges: 6–8 weeks, 10–14 weeks and 6–9 months. Ten male and 10 female mice for each age range were allocated, with the first five animals of each sex being used for haematology and the remaining five for biochemistry. Data from 19 health monitoring studies (2–3 studies/year) conducted between 2014 and 2023 were used in this retrospective analysis. These mice were housed in groups of five per IVC.

Specimen collection

Selected animals were fasted overnight (water ad libitum). Under isoflurane anaesthesia, animals were bled by retro-orbital plexus puncture by experienced personnel. Blood samples were taken from an initial five mice in each age group (450 µl/mouse) and placed in an anticoagulant tube (50 µl/vial, 2% EDTA), while blood collected from the other five mice (as much as 700 µl/mouse) was allowed to coagulate and serum was separated from it by centrifugation. Blood samples from each age group were collected by following the same procedure and mice were euthanized after completion of specimen collection.

Haematology and biochemistry analyte measurements

All samples were transported promptly to the inhouse Clinical Pathology Laboratory, which is accredited by NABL, India. Samples collected for clinical chemistry analysis were centrifuged at 4000 rev/min for 10 min at 24°C within 2 h of collection to harvest serum. CELL-DYN® 3700 and Cobas C311 have been our laboratory’s analysers for several years and have been well standardized and have documented traceability. Routine maintenance, calibration and quality control of the analyser were performed to ensure accuracy and precision. On the same day of collection, all samples were analysed within 5 h. Whole blood was used for the determination of haematology analytes: red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (PLT), white blood cell (WBC) and differential leucocyte count (neutrophil, lymphocyte, eosinophil, monocyte, basophil). The analyses were performed on the automated blood cell analyser CELL-DYN® 3700 System (Abbott, USA). Serum samples were used for biochemistry analytes: glucose (GLU), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), total bilirubin (TBIL), total protein (TP), albumin (ALB), urea (UREA) and creatinine (CREA). The analyses were performed using a Cobas C311 analyser (Roche Diagnostics, Switzerland). The analytes measured, their abbreviations and methods of analysis are presented in Table 1.

Table 1.

Abbreviations and methods of analysis of different haematological and biochemical parameters.

Analyte	Abbreviation	Method
White blood cell count	WBC	Laser light scatter^a
Red blood cell count	RBC	Light scattering – optical cytometer^a
Haemoglobin concentration	HGB	Cyanide-free haemoglobin methods^a
Haematocrit	HCT	Calculated
Mean corpuscular volume	MCV	Cumulative pulse height detection^a
Mean corpuscular haemoglobin	MCH	Calculated
Mean corpuscular haemoglobin concentration	MCHC	Calculated
Platelet	PLT	Light scattering – optical cytometer^a
Neutrophil	NEU	Flow cytometry^a
Neutrophil percentage	N%
Lymphocyte	LYMPH
Lymphocyte percentage	L%
Monocyte	MONO
Monocyte percentage	M%
Eosinophil	EOS
Eosinophil percentage	E%
Basophil	BASO
Basophil percentage	B%
Glucose	GLU	Hexokinase method^b
Aspartate aminotransferase	AST	IFCC method^b
Alanine aminotransferase	ALT
Alkaline phosphatase	ALP
Total bilirubin	TBIL	Colorimetric diazo method^b
Total protein	TP	Colorimetric biuret method^b
Albumin	ALB	Bromocresol green method^b
Urea	UREA	Kinetic method^b
Creatinine	CREA	Jaffe method^b

CELL-DYN® 3700 (Abbott).

Cobas C311 analyser (Roche Diagnostics, Switzerland).

Statistical analysis

Each reported data value was categorised by sex and age. Using SPSS, boxplot for each haematology and biochemistry analyte was visually checked for outliers, and significant outliers were identified as values below Q1 – 1.5 (interquartile range; IQR) or above Q3 + 1.5 (IQR) in accordance with Tukey’s method.¹⁴ Outliers were eliminated if biologically implausible, or if credible were included for further analysis. The Kolmogorov–Smirnov test was performed to determine the normality of the data distribution after elimination of significant outliers. Harris and Boyd’s test¹⁵ was used to determine combined or separated reference intervals (RIs) according to sex. Separate RI was calculated when z value > critical z value by robust method and if it did not demonstrate partition then combined RI was calculated using the non-parametric method according to age range by determinations of the 2.5th and 97.5th percentiles, as well as 90% confidence intervals of upper and lower limit of each RI. All computations were done in compliance with the CLSI’s guidelines¹⁶ and ASVCP’s guidelines.¹⁷ The number of animals, the corresponding mean, standard deviation, median, 95th percentile RIs, lower and upper limit at 90% confidence intervals for each analyte were used to express the results. To compare data between sexes (SPSS 21.0), independent-sample t-test (parametric) was used when conditions of normality were met. When normality test failed, the Mann–Whitney U test (non-parametric) was used. The differences linked to age for both sexes were performed by one-way analysis of variance (post-hoc analysis using with Tukey HDS test) using a statistical software program (SPSS 21.0). Differences were considered statistically significant at a p value of less than 0.05.

Results

Effect of sex and age on haematology analytes

The effect of sex and age on haematology analytes is shown in Figure 1. As expected, statistical comparisons and box plots indicated that sex and age had a significant impact on haematological and biochemical analytes. In 6–8-week-old mice (Table 2), male mice had significantly higher PLT, while female mice had significantly higher WBC, neutrophil (NEU) and lymphocyte (LYMPH). However, there were no significant differences between male and female mice in other reported analytes. Male mice had significantly greater neutrophil percentage (N%), while female mice had significantly higher LYMPH and lymphocyte percentage (L%) in mice aged 10–14 weeks (Table 3). Values for other reported analytes of 10–14-week-old mice did not demonstrate significant sex differences. In the case of 6–9-month-old mice (Table 4), male mice had significantly higher NEU and N%, while female mice had significantly higher RBC, HGB, HCT, PLT, LYMPH, L% and eosinophil percentage (E%). There were no significant sex differences observed in WBC, MCV, MCH, monocyte (MONO), monocyte percentage (M%), eosinophil (EOS), basophil (BASO) and basophil percentage (B%) values.

Figure 1.

Box plots show differences in haematology and biochemistry analytes of male and female ICR mice of different ages. WBC: white blood cell; RBC: red blood cell; HGB: haemoglobin; HCT: haematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; PLT: platelet; NEU: neutrophil; LYMPH: lymphocyte; MONO: monocyte; EOS: eosinophil; BASO: basophil; GLU: glucose; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphate; TP: total protein; ALB: albumin; CREA: creatinine; UREA: urea.

Table 2.

Reference intervals for haematology and biochemistry analytes in 6–8-week-old ICR mice.

Analyte	Sex	n	Mean	SD	Median	Reference interval	LRL 90% CI	URL 90% CI
RBC^a (10⁶/µl)	Male	94	8.3	0.51	8.2	7.43–9.17	6.91–7.56	9.03–9.29
RBC^a (10⁶/µl)	Female	94	8.3	0.37	8.2	7.43–9.17	6.91–7.56	9.03–9.29
HGB (g/dl)	Male	94	13.9	0.66	13.9	12.7–15.1	12.2–12.9	14.8–15.3
HGB (g/dl)	Female	93	14.0	0.51	14.0	12.7–15.1	12.2–12.9	14.8–15.3
HCT^a (%)	Male	94	47.9	3.19	47.8	41.7–54.4	40.0–42.8	53.3–54.8
HCT^a (%)	Female	94	47.8	3.08	47.8	41.7–54.4	40.0–42.8	53.3–54.8
MCV (fl)	Male	94	58.6	3.41	58.9	51.2–62.8	49.7–51.8	62.6–63.6
MCV (fl)	Female	94	57.8	3.41	58.4	51.2–62.8	49.7–51.8	62.6–63.6
MCH (pg)	Male	93	16.8	0.65	16.8	15.9–18.4	15.6–16	18.2–18.7
MCH (pg)	Female	93	17.0	0.67	16.8	15.9–18.4	15.6–16	18.2–18.7
MCHC (g/dl)	Male	94	29.0	1.44	29.0	26.9–31.8	26.5–27.2	31.6–32.1
MCHC (g/dl)	Female	94	29.4	1.43	29.4	26.9–31.8	26.5–27.2	31.6–32.1
PLT (10³/µl)	Male	94	997.4**	252.55	926.5	398–1445	320–485	1352–1556
PLT (10³/µl)	Female	91	885.8	201.60	819.0	397–1244	339–471	1166–1327
WBC (10³/µl)	Male	93	4.1	1.67	3.9	1.61–9.77	1.25–1.89	8.63–11.20
WBC (10³/µl)	Female	93	5.3**	2.26	5.1	1.61–9.77	1.25–1.89	8.63–11.20
NEU^a (10³/µl)	Male	83	0.60	0.21	0.57	0.27–1.42	0.17–0.31	1.29–1.60
NEU^a (10³/µl)	Female	93	0.80**	0.33	0.80	0.27–1.42	0.17–0.31	1.29–1.60
N%	Male	92	16.7	6.08	15.6	3.84–28.6	2.08–5.64	26.2–30.7
N%	Female	91	15.2	5.06	14.3	4.47–25.0	3.08–5.94	23.1–26.6
LYMPH (10³/µl)	Male	92	3.26	1.35	3.19	1.09–8.42	0.75–1.38	7.03–9.3
LYMPH (10³/µl)	Female	92	4.28**	1.92	4.11	1.09–8.42	0.75–1.38	7.03–9.3
L%	Male	92	79.6	6.01	80.0	67.0–90.2	63.0–68.6	88.8–92.1
L%	Female	91	81.0	5.46	81.2	67.0–90.2	63.0–68.6	88.8–92.1
MONO (10³/µl)	Male	90	0.04	0.03	0.03	0–0.13	0.001–0.006	1.22–0.14
MONO (10³/µl)	Female	90	0.05	0.04	0.04	0–0.13	0.001–0.006	1.22–0.14
M%	Male	91	1.06	0.68	0.96	0.11–3.02	0.03–0.17	2.80–3.22
M%	Female	93	1.09	0.81	0.94	0.11–3.02	0.03–0.17	2.80–3.22
EOS (10³/µl)	Male	93	0.07	0.07	0.04	0–0.24	0–0.003	0.23–0.25
EOS (10³/µl)	Female	90	0.07	0.07	0.04	0–0.24	0–0.003	0.23–0.25
E%	Male	94	1.50	1.31	1.05	0.07–4.43	0.03–0.09	4.10–5.60
E%	Female	90	1.33	1.38	0.70	0.07–4.43	0.03–0.09	4.10–5.60
BASO (10³/µl)	Male	90	0.03	0.02	0.03	0–0.16	0–0.002	0.12–0.19
BASO (10³/µl)	Female	89	0.05	0.05	0.03	0–0.16	0–0.002	0.12–0.19
B%	Male	93	0.88	0.73	0.74	0–2.74	0–0.05	2.35–3.35
B%	Female	93	0.99	0.88	0.84	0–2.74	0–0.05	2.35–3.35
GLU (mg/dl)	Male	89	53.6	17.63	49.6	25.5–94.1	18.2–29.6	84.7–115.0
GLU (mg/dl)	Female	87	56.4	17.81	52.5	25.5–94.1	18.2–29.6	84.7–115.0
AST (U/l)	Male	87	118.5	42.27	113.7	55.8–214.5	50.1–62.1	190.5–245.1
AST (U/l)	Female	92	116.1	39.00	105.9	55.8–214.5	50.1–62.1	190.5–245.1
ALT (U/l)	Male	87	41.9**	17.10	37.9	8.89–67.6	3.6–14.03	60.7–74.1
ALT (U/l)	Female	91	31.1	10.03	28.4	8.69–50.2	5.8–12.3	46.2–23.8
ALP (U/l)	Male	94	102.9	33.44	96.6	59.5–187.4	47.8–64.9	178.0–200.1
ALP (U/l)	Female	93	122.5**	31.96	115.5	59.5–187.4	47.8–64.9	178.0–200.1
TBIL (mg/dl)	Male	88	0.17	0.08	0.15	0.03–0.32	0–0.06	0.31–0.36
TBIL (mg/dl)	Female	85	0.15	0.06	0.15	0.03–0.32	0–0.06	0.31–0.36
TP (g/dl)	Male	93	5.5	0.31	5.5	4.87–6	4.7–4.9	6.0–6.2
TP (g/dl)	Female	95	5.5	0.32	5.5	4.87–6	4.7–4.9	6.0–6.2
ALB (g/dl)	Male	94	3.5	0.28	3.5	3.1–4.3	3.0–3.2	4.2–4.5
ALB (g/dl)	Female	95	3.7**	0.33	3.7	3.1–4.3	3.0–3.2	4.2–4.5
UREA^a (mg/dl)	Male	89	50.4	10.93	49.5	28.3–72.2	25.3–31.5	68.5–75.6
UREA^a (mg/dl)	Female	93	47.8	10.28	46.6	25.9–67.3	23.1–28.6	63.4–70.8
CREA (mg/dl)	Male	95	0.29	0.14	0.30	0.08–0.82	0.07–0.10	0.59–0.91
CREA (mg/dl)	Female	91	0.35	0.21	0.29	0.08–0.82	0.07–0.10	0.59–0.91

*p < 0.05, **p < 0.001: values differed significantly between male and female.

Statistical comparison based on parametric test.

LRL: lower reference limit; URL: upper reference limit; CI: confidence interval; RBC: red blood cell; HGB: haemoglobin; HCT: haematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; PLT: platelet; WBC: white blood cell; NEU: neutrophil; N%: neutrophil percentage; LYMPH: lymphocyte; L%: lymphocyte percentage; MONO: monocyte; M%: monocyte percentage; EOS: eosinophil; E%: eosinophil percentage; BASO: basophil; B%: basophil percentage; GLU: glucose; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphate; TBIL: total bilirubin; TP: total protein; ALB: albumin; UREA: urea; CREA: creatinine.

Table 3.

Reference intervals for haematology and biochemistry analytes in 10–14-week-old ICR mice.

Analyte	Sex	n	Mean	SD	Median	Reference interval	LRL 90% CI	URL 90% CI
RBC^a (10⁶/µl)	Male	93	8.4	0.48	8.4	7.57–9.32	7.21–7.69	9.18–9.60
RBC^a (10⁶/µl)	Female	93	8.4	0.36	8.4	7.57–9.32	7.21–7.69	9.18–9.60
HGB (g/dl)	Male	95	13.6	0.70	13.7	12.4–14.9	11.6–12.7	14.6–15.0
HGB (g/dl)	Female	91	13.8	0.46	13.8	12.4–14.9	11.6–12.7	14.6–15.0
HCT (%)	Male	95	46.8	2.81	46.9	41.2–51.8	38.8–42.4	51.3–52.6
HCT (%)	Female	93	47.2	2.77	47.5	41.2–51.8	38.8–42.4	51.3–52.6
MCV (fl)	Male	91	56.1	2.66	56.6	48.9–60.4	47.6–49.6	59.8–60.7
MCV (fl)	Female	94	55.9	3.14	57.0	48.9–60.4	47.6–49.6	59.8–60.7
MCH (pg)	Male	93	16.2	0.62	16.2	15.0–17.6	14.8–15.2	17.3–17.7
MCH (pg)	Female	92	16.4	0.60	16.5	15.0–17.6	14.8–15.2	17.3–17.7
MCHC^a (g/dl)	Male	95	29.1	1.41	29.1	26.8–32.3	26.4–27.1	31.9–32.8
MCHC^a (g/dl)	Female	93	29.3	1.49	29.3	26.8–32.3	26.4–27.1	31.9–32.8
PLT (10³/µl)	Male	88	947.6	193.35	892	492–1295	492–567	1222–1372
PLT (10³/µl)	Female	89	905.5	189.53	873	488–1257	439–546	1188–1330
WBC (10³/µl)	Male	93	4.1	1.58	3.8	1.59–7.89	1.02–1.74	7.39–8.40
WBC (10³/µl)	Female	93	4.4	1.73	4.2	1.59–7.89	1.02–1.74	7.39–8.40
NEU (10³/µl)	Male	86	0.76	0.31	0.70	0.19–1.49	0.003–0.286	1.41–1.63
NEU (10³/µl)	Female	91	0.70	0.37	0.60	0.19–1.49	0.003–0.286	1.41–1.63
N%	Male	85	19.5**	6.45	18.6	5.35–31.4	3.19–7.51	28.8–34.0
N%	Female	85	15.4	4.40	15.1	6.38–24.03	5.20–7.57	22.6–25.4
LYMPH (10³/µl)	Male	93	2.90	1.30	2.61	1.16–6.31	0.762–1.328	5.89–6.62
LYMPH (10³/µl)	Female	93	3.49**	1.37	3.42	1.16–6.31	0.762–1.328	5.89–6.62
L%	Male	88	74.9	8.36	76.6	54.9–87.5	51.5–64.8	86.6–92.0
L%	Female	87	80.2**	4.97	80.6	54.9–87.5	51.5–64.8	86.6–92.0
MONO (10³/µl)	Male	87	0.05	0.05	0.04	0–0.19	0.001–0.006	0.17–0.25
MONO (10³/µl)	Female	88	0.05	0.05	0.04	0–0.19	0.001–0.006	0.17–0.25
M%	Male	82	1.16	0.98	0.91	0.08–3.76	0.02–0.16	3.41–4.10
M%	Female	87	1.11	0.78	1.00	0.08–3.76	0.02–0.16	3.41–4.10
EOS (10³/µl)	Male	93	0.06	0.06	0.03	0–0.22	0–0.002	0.19–0.25
EOS (10³/µl)	Female	88	0.06	0.06	0.03	0–0.22	0–0.002	0.19–0.25
E%	Male	93	1.46	1.48	0.92	0.03–5.64	0–0.08	4.60–6.60
E%	Female	92	1.58	1.69	0.76	0.03–5.64	0–0.08	4.60–6.60
BASO (10³/µl)	Male	94	0.04	0.04	0.03	0–0.16	0–0.001	0.15–0.17
BASO (10³/µl)	Female	93	0.05	0.05	0.04	0–0.16	0–0.001	0.15–0.17
B%	Male	93	1.09	0.95	1.01	0–3.1	0–0.02	2.89–3.27
B%	Female	89	1.00	0.87	0.86	0–3.1	0–0.02	2.89–3.27
GLU^a (mg/dl)	Male	94	54.5	15.22	52.4	31.8–99.5	28.1–34.8	88.5–105.2
GLU^a (mg/dl)	Female	88	62.7**	15.67	60.6	31.8–99.5	28.1–34.8	88.5–105.2
AST (U/l)	Male	91	141.7**	59.20	133.5	17.5–256.3	0.95–34.5	235.2–275.6
AST (U/l)	Female	89	105.6	35.13	96.1	28.0–173.6	19.7–40.2	159.8–186.1
ALT (U/l)	Male	88	66.0**	44.05	49.1	15.0–191.9	11.6–17.7	158.5–230.8
ALT (U/l)	Female	86	30.6	9.76	29.0	9.1–48.6	5.1–13.3	44.1–53.0
ALP (U/l)	Male	95	57.3	17.88	56.0	29.8–119.9	25.1–33.3	109.8–135.3
ALP (U/l)	Female	92	78.1**	22.30	74.6	29.8–119.9	25.1–33.3	109.8–135.3
TBIL (mg/dl)	Male	95	0.22	0.09	0.21	0.02–0.40	0–0.07	0.36–0.45
TBIL (mg/dl)	Female	90	0.18	0.08	0.15	0.02–0.40	0–0.07	0.36–0.45
TP (g/dl)	Male	95	5.5	0.29	5.5	5.0–6.2	4.8–5.1	6.1–6.3
TP (g/dl)	Female	94	5.7**	0.25	5.7	5.0–6.2	4.8–5.1	6.1–6.3
ALB (g/dl)	Male	95	3.4	0.28	3.4	2.98–4.40	2.8–3.1	4.3–4.4
ALB (g/dl)	Female	95	3.8**	0.37	3.7	2.98–4.40	2.8–3.1	4.3–4.4
UREA^a (mg/dl)	Male	91	52.1**	10.21	51.0	30.4–71.4	27.0–31.1	67.5–74.9
UREA^a (mg/dl)	Female	90	44.6	7.65	44.0	28.9–59.6	26.8–30.8	57.0–62.0
CREA (mg/dl)	Male	95	0.28	0.14	0.31	0.08–0.76	0.07–0.09	0.62–0.93
CREA (mg/dl)	Female	91	0.34*	0.21	0.30	0.08–0.76	0.07–0.09	0.62–0.93

*p < 0.05, **p < 0.001: values differed significantly between male and female.

Statistical comparison based on parametric test.

Table 4.

Reference intervals for haematology and biochemistry analytes in 6–9-month-old ICR mice.

Analyte	Sex	n	Mean	SD	Median	Reference interval	LRL 90% CI	URL 90% CI
RBC^a (10⁶/µl)	Male	90	7.6	0.73	7.7	6.42–9.45	5.83–6.56	9.22–9.64
RBC^a (10⁶/µl)	Female	93	8.5**	0.54	8.5	6.42–9.45	5.83–6.56	9.22–9.64
HGB (g/dl)	Male	90	12.2	0.97	12.3	10.3–14.8	10.0–10.7	14.4–14.9
HGB (g/dl)	Female	87	13.5**	0.60	13.5	10.3–14.8	10.0–10.7	14.4–14.9
HCT^a (%)	Male	91	42.2	3.71	42.1	35.4–51.5	32.3–37.8	50.4–52.3
HCT^a (%)	Female	93	46.3**	3.10	46.3	35.4–51.5	32.3–37.8	50.4–52.3
MCV (fl)	Male	90	55.6	3.02	55.9	47.3–61.8	46.1–48.0	60.7–63.6
MCV (fl)	Female	94	54.8	3.90	55.4	47.3–61.8	46.1–48.0	60.7–63.6
MCH^a (pg)	Male	90	16.0	0.73	15.9	14.5–17.9	14.1–14.6	17.6–18.3
MCH^a (pg)	Female	94	16.1	0.96	16.0	14.5–17.9	14.1–14.6	17.6–18.3
MCHC^a (g/dl)	Male	92	28.8	1.24	28.9	26.9–32.1	26.6–27.1	31.4–32.4
MCHC^a (g/dl)	Female	94	29.4	1.39	29.3	26.9–32.1	26.6–27.1	31.4–32.4
PLT (10³/µl)	Male	90	1099.0	325.06	1010.5	712–1796	275–763	1741–2014
PLT (10³/µl)	Female	91	1172.0*	240.28	1135.0	712–1796	275–763	1741–2014
WBC (10³/µl)	Male	86	3.5	1.46	3.5	1.22–7.25	0.9–1.45	6.79–8.09
WBC (10³/µl)	Female	94	3.9	1.72	3.4	1.22–7.25	0.9–1.45	6.79–8.09
NEU (10³/µl)	Male	86	1.36**	0.68	1.30	0.30–2.78	0.19–0.36	2.32–3.23
NEU (10³/µl)	Female	93	0.95	0.44	0.89	0.30–2.78	0.19–0.36	2.32–3.23
N%	Male	93	39.7**	12.88	40.1	13.8–65.3	9.96–17.6	61.5–69.2
N%	Female	92	25.8	10.17	23.8	3.29–44.7	0.89–6.10	40.9–48.6
LYMPH (10³/µl)	Male	89	1.96	0.94	1.99	0.59–5.49	0.29–0.73	5.07–5.83
LYMPH (10³/µl)	Female	94	2.69*	1.41	2.29	0.59–5.49	0.29–0.73	5.07–5.83
L%	Male	93	54.1	13.98	54.5	30.7–84.9	20.7–34.6	81.9–87.4
L%	Female	92	67.7**	10.96	69.5	30.7–84.9	20.7–34.6	81.9–87.4
MONO (10³/µl)	Male	87	0.10	0.09	0.09	0–0.31	0.27–0.006	0.27–0.36
MONO (10³/µl)	Female	90	0.09	0.08	0.07	0–0.31	0.27–0.006	0.27–0.36
M%	Male	89	2.81	2.17	2.49	0.10–7.88	0–0.22	6.71–8.84
M%	Female	88	2.40	1.91	2.07	0.10–7.88	0–0.22	6.71–8.84
EOS (10³/µl)	Male	91	0.04	0.04	0.02	0–0.17	0–0.002	0.14–0.21
EOS (10³/µl)	Female	87	0.05	0.05	0.02	0–0.17	0–0.002	0.14–0.21
E%	Male	86	0.90	1.00	0.41	0.04–4.60	0–0.06	4.1–5.9
E%	Female	90	1.44*	1.51	0.75	0.04–4.60
BASO (10³/µl)	Male	86	0.04	0.04	0.03	0–0.17	0–0	0.16–0.20
BASO (10³/µl)	Female	90	0.05	0.05	0.04	0–0.17	0–0	0.16–0.20
B%	Male	90	1.24	1.13	1.15	0–4.48	0–0	4.21–5.04
B%	Female	92	1.51	1.36	1.12	0–4.48	0–0	4.21–5.04
GLU^a (mg/dl)	Male	92	69.0	24.38	66.0	32.3–119.6	17.4–36.1	113.2–144.5
GLU^a (mg/dl)	Female	92	74.2	22.16	72.5	32.3–119.6	17.4–36.1	113.2–144.5
AST (U/l)	Male	92	174.7**	84.77	155.6	59.9–402.9	52.7–68.0	347.7–459.3
AST (U/l)	Female	91	117.9	32.93	116.9	49.3–180.9	40.3–58.3	169.5–192.1
ALT (U/l)	Male	88	70.9**	44.17	57.7	17.9–186.6	15.4–21.1	154.3–223.2
ALT (U/l)	Female	86	44.7	15.75	43.6	11.2–74.3	6.7–16.0	68.5–80.2
ALP^a (U/l)	Male	94	38.6	13.99	36.8	18.8–89.3	16.6–21.9	82.3–106.6
ALP^a (U/l)	Female	92	56.0**	17.45	54.0	18.8–89.3	16.6–21.9	82.3–106.6
TBIL (mg/dl)	Male	91	0.21	0.09	0.18	0.046–0.37	0.01–0.08	0.36–0.44
TBIL (mg/dl)	Female	91	0.18	0.08	0.15	0.046–0.37	0.01–0.08	0.36–0.44
TP (g/dl)	Male	94	5.5	0.39	5.5	4.8–6.5	4.7–5.0	6.3–6.7
TP (g/dl)	Female	93	5.8**	0.34	5.8	4.8–6.5	4.7–5.0	6.3–6.7
ALB (g/dl)	Male	95	3.2	0.29	3.2	2.78–4.1	2.5–2.8	4.1–4.2
ALB (g/dl)	Female	95	3.6**	0.38	3.5	2.78–4.1	2.5–2.8	4.1–4.2
UREA (mg/dl)	Male	93	48.1**	12.50	47.0	22.3–72.4	19.1–25.8	68.5–76.3
UREA (mg/dl)	Female	94	41.8	8.46	41.0	23.8–57.8	21.8–26.1	54.7–61.0
CREA (mg/dl)	Male	94	0.28	0.15	0.28	0.07–0.69	0.04–0.09	0.58–0.09
CREA (mg/dl)	Female	91	0.33	0.19	0.36	0.07–0.69	0.04–0.09	0.58–0.09

*p < 0.05, **p < 0.001: values differed significantly between male and female.

Statistical comparison based on parametric test.

Age-related differences in haematology analytes are presented in Table 5 for both sexes. In relation to RBC, HGB and HCT mean values (Tables 2 –4), there were no statistically significant differences between sexes up to 14 weeks of age, while these values were significantly higher in 6–9-month-old females. These analytes had a statistically significant effect of age as shown in Table 5 and Figure 1. MCV and MCH mean values had no sex effect but had a significant effect of age. MCHC values were similar between age ranges and sexes. PLT mean values showed statistically significant differences according to age and were found to be higher in older mice; however, statistically significant sex differences were observed in 6–8-week-old and 6–9-month-old mice. Regarding the WBC, sex differences were observed in 6–8-week-old females compared with other groups; however, significant effect of age was observed only in females (Table 5). In the case of differential absolute leucocyte counts, there were statistically significant differences between sexes for LYMPH in all age ranges and also an age effect. NEU counts had a significant effect of age and increased with age; however, sex differences were observed in 6–8-week-old and 6–9-month-old mice. MONO, EOS and BASO counts had no sex differences, but an age effect was observed in both sexes for MONO. Regarding percentage values, N% and L% had a significant effect according to age (Table 5), while a significant sex effect was observed in 10–14-week-old and 6–9-month-old mice. There were no significant sex differences in M%, E% and B% except for E% in 6–9-month-old mice; however, there was a significant effect of age in M% and B% in both sexes.

Table 5.

Age-related mean values of haematological and biochemical analytes for male and female ICR mice.

Analyte	Male			Female
Analyte	6–8 weeks	10–14 weeks	6–9 months	6–8 weeks	10–14 weeks	6–9 months
RBC (10⁶/µl)	8.3^C	8.4^B	7.6	8.3	8.4^a	8.5^c
HGB (g/dl)	13.9^C	13.6^B	12.2	14.0^a,C	13.8^B	13.5
HCT (%)	47.9^C	46.8^B	42.2	47.8^c	47.2	46.3
MCV (fl)	58.6^A,C	56.1	55.6	57.8^a,C	55.9	54.8
MCH (pg)	16.8^A,C	16.2	16.00	17.0^A,C	16.4^b	16.1
MCHC (g/dl)	29.0	29.1	28.8	29.4	29.3	29.4
PLT (10³/µl)	997.4	947.6	1099^B,c	885.8	905.5	1172^B,C
WBC (10³/µl)	4.1	4.1	3.5	5.3^a,C	4.4	3.9
NEU (10³/µl)	0.60	0.76	1.36^B,C	0.80	0.70	0.95^B,c
N%	16.7	19.5	39.7^B,C	15.2	15.4	25.8^B,C
LYMPH (10³/µl)	3.26^C	2.90^B	1.96	4.28^a,C	3.49^b	2.69
L%	79.6^a,C	74.9^B	54.1	81.0^C	80.2^B	67.7
MONO (10³/µl)	0.04	0.05	0.10^B,C	0.05	0.05	0.09^B,C
M%	1.06	1.16	2.81^B,C	1.09	1.11	2.40^B,C
EOS (10³/µl)	0.07^c	0.06^b	0.04	0.07	0.06	0.05
E%	1.50^c	1.46^b	0.90	1.33	1.58	1.44
BASO (10³/µl)	0.03	0.04^a	0.04	0.05	0.05	0.05
B%	0.88	1.09	1.24^c	0.99	1.00	1.51^b,c
GLU (mg/dl)	53.6	54.5	69.0^B,C	56.4	62.7	74.2^B,C
AST (U/l)	118.5	141.7	174.7^b,C	116.1	105.6	117.9
ALT (U/l)	41.9	66.0^A	70.9^C	31.1	30.6	44.7^B,C
ALP (U/l)	102.9^A,C	57.3^B	38.6	122.5^A,C	78.10^B	56.0
TBIL (mg/dl)	0.17	0.22	0.21	0.15	0.18	0.18
TP (g/dl)	5.5	5.5	5.5	5.5	5.7^A	5.8^b,C
ALB (g/dl)	3.5^C	3.4^B	3.2	3.7^c	3.8^B	3.6
UREA (mg/dl)	50.4	52.1^b	48.1	47.8^a,C	44.6	41.8
CREA (mg/dl)	0.29	0.28	0.28	0.35	0.34	0.33

Superscript lettering A, B, C indicates statistically significant differences at p < 0.001, and superscript lettering a, b, c indicates statistically significant at p < 0.05.

Significant difference 6–8 weeks vs. 10–14 weeks.

Significant difference 10–14 weeks vs. 6–9 months.

Significant difference 6–8 weeks vs. 6–9 months.

RBC: red blood cell; HGB: haemoglobin; HCT: haematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; PLT: platelet; WBC: white blood cell; NEU: neutrophil; N%: neutrophil percentage; LYMPH: lymphocyte; L%: lymphocyte percentage; MONO: monocyte; M%: monocyte percentage; EOS: eosinophil; E%: eosinophil percentage; BASO: basophil; B%: basophil percentage; GLU: glucose; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphate; TBIL: total bilirubin; TP: total protein; ALB: albumin; UREA: urea; CREA: creatinine.

Effect of sex and age on biochemistry analytes

The effects of sex and age on biochemistry analytes are presented in Figure 1. Sex differences for 6–8-week-old mice are summarized in Table 2. Female mice had significantly higher ALP and ALB, while male mice had significantly higher ALT. There were no significant differences found between sexes in GLU, AST, TP, UREA and CREA values. The data for 10–14-week-old mice are presented in Table 3. Male mice had significantly higher AST, ALT and UREA, while female mice had significantly higher GLU, ALP, TP, ALB and CREA. In the age group 6–9 months, biochemistry analytes of both sexes are depicted in Table 4. Male mice had significantly higher AST, ALT and UREA, while female mice had significantly higher ALP, TP and ALB. There were no significant differences found between sexes in GLU and CREA values.

Age-related differences for biochemistry analytes are presented in Table 5 for both sexes. In relation to age, 6–9-month-old male mice had significantly higher GLU, AST, ALT and TP, whereas female mice had significantly higher GLU, ALT and TP. Mice 6–8 weeks old had significantly higher ALP in both sexes. Males had significantly higher ALT, whereas females had significantly higher ALP at all age ranges (Tables 2 –4). AST and TP values had significant sex differences in 10–14-week-old and 6–9-month-old mice compared with 6–8-week-old mice. Regarding the mean values of ALB, there was a significant effect of sex and it was found to be higher in female mice at all age ranges. UREA had significant effect according to age in both sexes; however, significant sex differences were observed in 10–14-week-old and 6–9-month-old mice. Significant sex differences for GLU and CREA were found only in 10–14-week-old mice compared with other groups. CREA had no significant effect of age; however, GLU had significant effect of age in both sexes. TBIL concentrations did not reach the detection limit in most animals and were not subjected to further statistical analysis.

Discussion

Mice are the most commonly used animals in life sciences studies. Pathological processes may influence metabolism and alter the results obtained in experimental procedures. Therefore, knowledge of normal values for common haematological and biochemical analytes is important.¹⁸ Thus, we have characterized the ICR mouse data from 6–8 weeks to more than six months of age for both males and females. All analytes did not need sex partitioning according to results of the Harris and Boyd test, whereas PLT, N%, AST, ALT and UREA need sex partitioning. When the results obtained in this investigation were compared with the published papers and supplier data, we observed similarities and differences among measured haematological and biochemical values. There are several factors that may influence the results of analytes, such as sex, age, site of blood collection, number of samples analysed, environmental conditions, instrument, reagent and method of determination, et cetera.

Haematological analytes are used as biomarkers in the diagnosis of organ or tissue injuries and other pathologies. In this study, we established a RI of haematology and biochemistry analytes from healthy ICR mice at different age ranges. In clinical practice, RBC, HCT, HGB, MCV, MCH and MCHC values are commonly used to evaluate the erythrogram.¹⁹ We observed age-related declines in HGB, HCT, MCV and MCH concentrations as age increased (Table 5); however, sex differences were found for HGB and HCT in 6–9-month-old mice compared with other groups. Other authors^11,12 and suppliers^20
–22 reported similar values for HGB and MCH. In comparison with the results (6–14 weeks), Serfilippi et al.¹² reported lower HCT (range: 40–48%) and MCV (range: 42–48 fl) values, while Shin et al.¹¹ reported higher HCT mean values (50–63%). As noted, RBC concentration was found to be similar in both sexes up to 14 weeks of age, but 6–9-month-old females displayed significantly higher concentration than males. RBC mean values (8–8.4 10⁶/µl) obtained in the current study were lower than those reported (9–10 10⁶/µl) by other authors^11,12 and suppliers.^20
–22

Platelets are anucleate fragmented cells derived from megakaryocytes. They have haemostatic (buffer) functions, and maintain endothelium integrity by releasing pro-angiogenic cytokines.²³ These cells can be activated spontaneously or in response to stimuli, depending on lineage activation stage.²⁴ In this study, we identified significant sex differences in PLT counts for mice aged 6–8 weeks and 6–9 months, but no significant difference for mice aged 10–14 weeks. There was an age effect and older mice had significantly higher level of PLT than younger mice. PLT readings were found to be less than reported by others.^11,12,20,21 Serfilippi et al.¹² reported PLT mean values (1112–1207 10³/µl) for 17-week-old mice and it was found in the range 1000–1500 10³/µl for 8–10-week-old mice as per supplier data.^20,21 Platelet production can increase due to inflammatory disease, myeloproliferative disease, neoplasia and iron deficiency.^25
–27

Leukocytes participate in immune and inflammatory processes, being responsible for mediating innate and adaptive immune responses. In this study, WBC progressively decreased with age in both sexes, but significant effect of age was observed only in females. Our results were similar to those of Shin et al.,¹¹ who reported higher WBC counts in males, but we found higher in females. In comparison to the results, Serfilippi et al.¹², Charles River Laboratories (CRL)²⁰ and Inotiv²² reported higher WBC counts (7–13 10³/µl). The absolute counts of NEU increased while LYMPH decreased in relation to age; however, percentage counts of N%, M% and B% increased, whereas L% decreased with age (Table 5). Serfilippi et al.¹² reported higher absolute counts (NEU 2.08, LYMPH 8.3, MONO 0.24, EOS 0.25 10³/µl, age: 17 weeks) and CRL²⁰ also reported higher counts (NEU 2.02, LYMPH 5.73, MONO 0.58, EOS 0.21 10³/µl, age: 8–10 weeks) compared with reported results. CRL²⁰ reported higher percentage counts for 8–10-week-old mice (N% 23.45%, M% 6.77%, E% 2.16%) but lower percentage counts for L% and B% (L% 67.2%, B% 0.41%). Schwab et al.²⁸ report that lymphocyte counts can decrease with handling or other stressors and with age in mice and Provencher Bolliger et al.²⁵ report that NEU counts increase with age. Generally, BASO counts are normally low and quite variable in rats and mice.²⁹

The determination of biochemical analytes provides important information on clinical status, nutritional balance and the metabolic functioning of the organs and tissue, as well as evidence of occult diseases, enabling the monitoring of treatment and prognosis.^30,31 Clinical evaluation of renal function is based on measurement of UREA, together with CREA.^32,33 UREA is the main nitrogen metabolite derived from protein degradation;³³ 90% is excreted by the kidneys, ∼40–70% returns to plasma.³² ICR mice had an average CREA concentration of 0.28–0.35 mg/dl, which was consistent with other authors,^11,12 and there was no significant difference between age ranges. In mice aged 10–14 weeks and 6–9 months, males had significantly higher UREA compared with females. There was no significant difference according to age in males, while it was observed in females, and its value decreased as age increased.

The assessment of hepatic function in response to anatomical or biochemical alterations³⁴ is commonly subjected to dosage testing of ALT, AST and ALP.³⁵ Age and sex had a significant effect on ALT and concentration increased with age. Males had noticeably higher concentrations at all age ranges, and similar values for 6–14-week-old mice were reported by authors.^11
–13,20 AST was found to be significantly higher in 10–14-week-old and 6–9-month-old males, and, as mice aged, its concentration gradually increased. The results of this study were higher than those reported by authors.^11,12,20,22 ALP was significantly higher in females in all age ranges, and its concentration decreased as age increased. The reported values are in line with authors.^12,13 However, Shin et al.¹¹ and CRL²⁰ reported higher ALP mean values (200–300 U/l, 136–156 U/l respectively) in 8–12-week-old mice.

The evaluation of TP is an important determinant of metabolism homeostasis.³⁶ Proteins are found in all components of cells, being fundamental to their structures and functions.³⁷ The concentration of TP is highly related to symptoms such as weight loss.³⁸ Mice aged 10–14 weeks and 6–9 months had significant sex differences and were found to have higher concentrations in females than males. There was significant effect of age in females and ALB indices increased as age increased, while it was not observed in males. ALB was significantly higher in females in all age ranges and its concentration decreased with age. The reported TP and ALB values are similar to those reported by authors^11,12 and suppliers.^20
–22 Naturally, the concentration of glucose in blood is tightly regulated via homeostasis.³⁹ With the exception of mice aged 10–14 weeks, our findings suggest that there were no significant sex differences for GLU, but there was a significant age effect. The GLU levels of overnight-fasted mice are expected to decrease, so it was difficult to compare values with published data.

Our vivarium is well-established, environmental parameters are maintained uniformly throughout the period of studies and in general, and monitored strictly for probable variables at the required frequency. The staff handling the animals are well trained, experienced and unique. Hence, there is a minimum possibility of the effect of variables on the outcome of results. So the longer duration of study should not have any impact on the final outcome of the study. In the present study, we determined separated sex RIs from a limited sample size (<120); however, combined RIs calculated from sample sizes of more than 120 could be an addition to the available literature. In addition, more accurate data could be obtained by establishing RIs for both sexes using a larger sample size. When evaluating the reference values provided in this article, researchers should consider factors impacting haematology and biochemistry analytes.

Conclusions

The goal of this study was to generate a RI for common haematology and biochemistry analytes of ICR mice in three periods of a one-year life span. As a result, we have generated and presented the normal haematology and biochemistry analytes of healthy ICR mice of both sexes at three different age ranges. The RIs presented here will be used by investigators for study design, comparing data and clinical assessment when ICR mice are used as a model.

Supplemental Material

sj-pdf-1-lan-10.1177_00236772241260909 - Supplemental material for Establishment of reference intervals of haematology and biochemistry analytes in ICR mice of different ages

Supplemental material, sj-pdf-1-lan-10.1177_00236772241260909 for Establishment of reference intervals of haematology and biochemistry analytes in ICR mice of different ages by Suresh Patel, Satish Patel, Ashvin Kotadiya, Samir Patel, Bhavesh Shrimali, Tushar Patel, Harshida Trivedi, Vishal Patel, Jogeswar Mahapatra and Mukul Jain in Laboratory Animals

Footnotes

Acknowledgement

The authors wish to express their gratitude to the management at Zydus Research Centre, Zydus Lifesciences Ltd, Ahmedabad, India.

Data availability statement

All pertinent information is contained in the manuscript, and the corresponding author can provide original and derived data that support the findings of this work upon request by emailing suresh.g.patel@zyduslife.com.

Declaration of conflicting interests

The authors have no conflicts of interest to declare.

Ethical approval statement

All health monitoring studies were approved by the Institutional Animal Ethics Committee of Zydus Research Centre, Zydus Lifesciences Ltd, Ahmedabad, India.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Suresh Patel

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