Effects of Weekly Blood Collection in C57BL/6 Mice

Animals and housing.

All mice used during this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals¹² at the University of Illinois at Chicago (Chicago, IL), an AAALAC-accredited institution. All procedures were reviewed and approved by the University of Illinois at Chicago Animal Care Committee. Male and female C57BL/6 mice were purchased from Charles River Laboratories (Portage, MI). Mice were maintained in facilities in which sentinel mice tested negative for the following agents: Sendai virus, pneumonia virus of mice, mouse hepatitis virus, minute virus of mice, mouse parvovirus, Theiler murine encephalomyelitis virus, reovirus, rotavirus, mouse adenovirus, polyoma virus, K virus, mouse cytomegalovirus, mouse thymic virus, lymphocytic choriomeningitis virus, hantavirus, ectromelia virus, lactate dehydrogenase elevating virus, mouse norovirus, Mycoplasma pulmonis, and Helicobacter spp. In addition, sentinel mice were free of helminths and external parasites. Mice were housed 3 to 5 per cage by sex in static autoclaved (sterilized) polysulfone microisolation cages (Ancare, Bellmore, NY) with irradiated diet (7912, Harlan Teklad, Madison, WI), autoclaved municipal water in bottles, and autoclaved corncob bedding (7090, Harlan Teklad) with a 14:10-h light:dark cycle. Mice were allowed to acclimate for at least 7 d prior to the study. At the start of experiments, mice ranged from 10 to 14 wk of age and weighed an average of 25.9 g and 20.2 g for male and female mice, respectively.

Experimental design.

All mice in this study had a percentage of their TBV collected once weekly (every 7 d) for 6 wk. Initially, a group of 33 male and 35 female mice had 15% of their TBV collected weekly. The data obtained from this group indicated that mice could recover each week from this level of weekly blood collection. A pilot study then was conducted with 4 male and 5 female mice at 20% TBV and 11 male and 6 female mice at 25% TBV. The data from this pilot study indicated that female mice in both the 20% and 25% TBV collection groups and male mice in the 20% TBV collection group could recover each week from this level of weekly blood collection. Findings showed that male mice did not adequately recover from a weekly 25% TBV collection. In light of these data, 19 additional male mice were bled at the 20% TBV level, and 18 additional female mice were bled at the 25% TBV level.

Sedation and blood collection.

Mice were weighed immediately prior to each blood collection. The amount of blood collected at each time point was determined given the assumption that TBV was 6% of body weight.^2,15,17 Mice were sedated with fentanyl citrate (0.8 mg/kg IP; Hospira, Lake Forest, IL) 1 to 5 min prior to blood collection. The volume of fentanyl injected in the 15% TBV group was approximately 1.8 times the amount of blood that was collected. Fentanyl in the 20% and 25% TBV groups was diluted with 0.9% sodium chloride (Baxter Healthcare Corporation, Deerfield, IL) so that the final volume administered was equal to approximately 1.8 times the volume of blood that was removed. Blood was collected from the retroorbital sinus in aliquots of 70 µL by using 70-µL nonheparinized microhematocrit capillary tubes (Fisher Scientific, Pittsburgh, PA). The last tube in each collection was filled in a proportional manner based on the length of tube needed to meet each animal's respective blood collection requirement. We estimated samples collected were within 6% of the calculated blood volume.

Blood collection tubes.

Blood collection tubes were prepared inhouse, because some of the volumes collected were less than that recommended for commercially available anticoagulant tubes. A solution of 18 mg K₂EDTA (Fisher Scientific, Pittsburgh, PA) per 1 mL of distilled water was prepared. From this 18 mg/mL solution, 14-µL aliquots were pipetted into 0.5 mL microcentrifuge tubes (Fisher Scientific), which then served as the blood collection tubes. Prior testing demonstrated that this concentration of EDTA did not affect hematocrit values or cause RBC crenation.

Sample processing.

For some of the female mice in the 15% TBV group, the volume of blood collected was less than the 200 µL required by the hematology analyzer used for this study, necessitating dilution of the samples prior to analysis. All samples from all mice were diluted prior to analysis even if more than 200 µL blood was collected. During blood collection, 140 µL of the total volume collected from each mouse was placed into a blood collection tube (described previously) for a final ratio of 1.8 mg K₂EDTA per 1 mL blood.¹³ The remainder of the collected blood was discarded. To dilute the samples, 110 µL of anticoagulated blood was transferred to another 0.5-mL microcentrifuge tube containing 110 µL diluent (Sheath Rinse for Advia 120, Siemens Healthcare Diagnostics, Tarrytown, NY) for a final sample volume of 220 µL (1:2 dilution). The hematology analyzer was programmed to account for the dilution factor.

Hematologic analyses.

Analyses were performed on an automated hematology analyzer (Advia 120, Siemens Healthcare Diagnostics) using mouse-specific algorithms and parameters (Technicon H•1E MultiSpecies Software, version 3.0, Siemens Healthcare Diagnostics). Parameters determined included WBC count and differential, RBC count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, and reticulocyte count and percentage.

Animal monitoring.

Mice in all groups were observed daily for lethargy or other signs of illness. Mice in the 15% TBV groups were weighed weekly immediately prior to each blood collection. Mice in the 20% and 25% TBV groups were weighed immediately prior to and at 1, 2, and 5 d after each blood collection.

Endpoint criteria.

Mice with a hematocrit below 40% for 2 consecutive weeks were euthanized, and a complete necropsy was performed.

Statistical analyses.

A generalized estimating equation model was done on all parameters for male and female mice of all groups, comparing mean values from collections 2 through 6 with mean baseline (collection 1) values by using SAS 9.2 software (SAS Institute, Cary, NC). Results were considered statistically significant at P ≤ 0.01. The 95% confidence intervals were determined for mean hemoglobin values from collections 2 through 6 for the 15% TBV male and female, 20% TBV male, and 25% TBV female groups and compared with mean baseline values.

Baseline hemoglobin and anemia.

Baseline hemoglobin values (mean ± 2 SD) for each sex were calculated by using data from the first blood collection time point from all mice in all groups (n = 67 male and 64 female mice). Anemia was defined as a hemoglobin concentration more than 2 SD below the mean of baseline hemoglobin values.

Morbidity and mortality.

Mice in all groups remained healthy throughout the course of the study. Overall, animals exhibited normal behavior, and no signs of illness or lethargy were noted. Immediately after blood collection, no ill effects from a behavioral perspective were seen in any of the mice in the 15% and 20% TBV male groups or the 15% and 25% TBV female groups. In contrast, mice in the 25% TBV male pilot group took longer to stand and ambulate after blood collection, when compared with mice in other groups.

Necropsy.

Only one mouse reached endpoint criteria and was euthanized. A complete necropsy was performed on this mouse from the 25% TBV male pilot group. No gross abnormalities were seen, and there were no histopathologic changes that would account for the low hematocrit values. It was concluded that the low values were a direct result of the blood collection procedures.

15% and 20% TBV male groups and 15% and 25% TBV female groups.

The mean body weights of male and female mice in the 15% TBV groups, male mice in the 20% TBV group, and female mice in the 25% TBV group increased over the course of the study (Figure 1). Hematocrit, RBC count, hemoglobin concentration, mean corpuscular volume, and reticulocyte values (mean ± 2 SD) from each blood collection are presented in Table 1. Hemoglobin values (mean ± 1 SD) from each blood collection are presented graphically in Figure 2. The 95% confidence intervals for hemoglobin concentration (g/dL) were 14.0 to 14.5 for 15% TBV male mice, 14.6 to 14.9 for 15% TBV female mice, 13.1 to 13.7 for 20% TBV male mice, and 14.3 to 14.7 for 25% TBV female mice.

20% female pilot group.

The results demonstrated that female mice (n = 5) can recover hematologically from weekly collection of 20% TBV (data not shown).

25% male pilot group.

Over the course of the study, the mean body weight of these mice increased from 25.0 g at baseline to 26.4 g at collection 6. However, 4 of 11 animals had a decreased weight at 1 wk after a blood collection for at least one collection time point during the study. For hematocrit, RBC count, and hemoglobin concentration, the mean values from collections 2, 3, 4, and 6 were statistically lower than the mean baseline values. No hematologic data was available from this group for collection 5, due to technical issues with the hematology analyzer. In one mouse, hematocrit values fell below 40% for 2 consecutive weeks, meeting our endpoint criteria. In light of these observations, we determined that male mice did not recover hematologically from weekly blood collection of 25% TBV. As this was a pilot study with only 11 animals, data is not shown.

Baseline hemoglobin and anemia.

Baseline hemoglobin values (mean ± 2 SD) in male and female mice were 15.0 ± 1.4 and 14.9 ± 1.0 g/dL, respectively, and the normal hemoglobin concentration ranges were 13.6 to 16.4 and 13.9 to 15.9 g/dL for male and female mice, respectively. Hemoglobin values less than 13.6 g/dL were considered anemic for male mice, whereas values less than 13.9 g/dL were considered anemic for female mice.

Anemia categorization.

An anemia severity scale based on hemoglobin was developed for our mice using an anemia categorization chart established by the World Health Organization.⁸ The mouse scale was created by calculating the percentage change from normal for the various anemic categories and applying this information to our mouse values (Table 2).

The percentages of hemoglobin concentration values from blood collections 2 through 6 that fell into the various anemia categories are presented in Table 3.

Other hematologic parameters.

No trends or patterns were noted for any TBV group in regard to WBC count or differential, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, and mean platelet volume (data not shown).