Determination of RBC Survival in C57BL/6 and C57BL/6-Tg(UBC–GFP) Mice

Abstract

Although several methods for determining erythrocyte lifespan are used in research studies that involve mice, all involve the alteration of RBC to allow for its tracking over time, which may affect overall RBC survival. The aims of this study were to determine 1) whether sex affects RBC survival; 2) whether RBC survival differs between the biotin method and an alternative method that uses GFP; and 3) whether repeat exposure of mice to biotin results in an antibiotin antibody response or decreased RBC survival. The results suggest no difference in the RBC half-life between male and female C57BL/6 mice (22.9 ± 1.2 and 22.4 ± 0.9 d, respectively). In addition, RBC half-life did not differ between the biotin- and GFP-based methods (20.5 ± 2.1 d and 22.7 ± 2.1 d, respectively). Finally, retransfusion of mice 90 d after an initial transfusion with biotin-labeled RBC did not induce detectable antibiotin antibodies nor alter the half-life of transfused biotin-labeled RBC (initial transfusion, 22.0 ± 1.2 d; subsequent transfusion, 23.4 ± 1.4 d, respectively).

RBC lifespan and senescence are important parameters used both clinically and in research studies of hereditary disorders of erythrocyte metabolism, transfusion medicine, and sepsis.^{8,21,27,32,35} Labeling RBC with a biotinylating reagent is a common method used to determine their circulating lifespan. Other methods involve using radioactive isotopes, such as ⁵¹Cr and ⁵⁹Fe.^7,20 Biotinylating reagents are preferred for various research applications with humans,^8,23,24 and are used in a variety of animal models.^{1,25,33,34,37} Once biotin attaches to RBC surface proteins, streptavidin (a protein derived from Streptomyces avidinii) that is labeled with a fluorescent dye is used to form a strong and rapid complex with biotin, thereby allowing for its detection through flow cytometry. Blood samples analyzed sequentially over a period of weeks will show a linear decline in biotin–streptavidin signal as labeled cells age and are cleared from the circulation through the reticuloendothelial system.

The characteristics of an ideal label for performing RBC survival studies include: 1) stable presence on or within the cell throughout its normal lifespan; 2) specificity for RBC; 3) inertness, such that the cell does not become prone to accelerated destruction; 4) nonrecycling (that is, the label does not reenter the circulation and bind to new cells after destruction of the labeled RBC); and 5) easy and accurate measurement by using available assays. Radioactive isotopes and other labels fulfill several of these criteria, but their limitations include elution from RBC as well as safety concerns.^7,22 In contrast, biotin poses little to no risk of accumulation or toxicity. The sulfo- N-hydroxysuccinimide–biotin ester used for RBC tracking studies in humans and animals can be administered directly or through the transfusion of biotinylated RBC. Although it is generally accepted that biotinylation of RBC does not affect their function, antibodies to biotin have been demonstrated in some human studies, posing the question of whether repeated administration of biotin ester or biotinylated RBC could interfere with subsequent results within the same subject.^4,20 Repeat transfusions of biotinylated RBC to mice have not been described in the literature. One aim of this study was to determine whether exposure to biotinylated RBC induces an antibiotin antibody response in mice. Furthermore, we tested whether the survival of biotinylated RBC changed after repeat exposure.

Recently GFP-expressing RBC have been used to track the posttransfusion survival and recovery of stored RBC administered to nonGFP-expressing recipient mice.^9,12,36 The C57BL/6-Tg(UBC–GFP)30Scha/J mouse strain is characterized by GFP expression under the control of a human ubiquitin C promoter. All tissues of these mice express GFP, including blood.²⁶ GFP expression appears to be consistent throughout life and does not otherwise alter the normal structure, physiology, or function of RBC. In addition, GFP is unaffected by ambient light contamination or degradation, drawbacks that are associated with fluorescent dyes.¹⁵ In addition, GFP allows for the separation of cell populations through flow cytometry.^9,11 Many qualities of GFP suggest that it may serve as a useful surrogate marker in place of other labeling techniques in mice. Therefore, we sought to evaluate the utility of UBC–GFP transgenic mice as an alternative to labeling RBC with biotin esters. Our aim for this work was to determine the survival of RBC in wild-type C57BL/6 mice and in the UBC–GFP strain and to compare methods for determining RBC half-life (T_1/2).

Materials and Methods

Animals.

Male and female C57BL/6 mice (age, 6 to 8 wk) were purchased from Charles River Laboratories (Wilmington, MA), and male and female C57BL/6-Tg(UBC-GFP)30Schaa/J mice (age, 6 to 8 wk) were acquired from Jackson Laboratories (stock no., 004353; Bar Harbor ME). Mice were housed in compatible single-sex groups within static microisolator cages, provided with free choice standard rodent chow diet (Purina Picolab 5058; LabDiet, St Louis, MO), autoclaved tap water, and autoclaved corncob bedding, cotton nesting material, and environmental enrichment (Shepherd Shacks, Shepherd Specialty Papers, Watertown, TN). Animal protocols were approved by the IACUC of Columbia University (New York, NY), and all rodent health monitoring, husbandry, and experimental procedures were performed in compliance with the recommendations of the Guide for the Care and Use of Laboratory Animals.¹⁶ The animal facility is AAALAC-accredited and maintained at an SPF status (free of mouse adenoviruses, mouse parvoviruses, mouse hepatitis virus, Theiler murine encephalomyelitis virus, mouse rotavirus, Sendai virus, pneumonia virus of mice, mouse reovirus type 3, lymphocytic choriomeningitis virus, ectromelia virus, Hantaan virus, Mycoplasma pulmonis, and endo- and ectoparasites).

Biotinylation and RBC transfusions.

EZ-Link Sulfo-NHS Biotin (Pierce, Rockford, IL) was prepared in the laboratory for intravenous injection. The powder was dissolved in sterile PBS, vortexed, and passed through a 0.22-µm filter and diluted to a final concentration of 1 mg biotin per 300-µL injection. Mice received the biotin reagent solution intravenously by retroorbital injection under anesthesia.

For transfusions of biotinylated RBC, 2 male donor mice of the selected strain received the biotinylating reagent as described earlier. At 2d after injection, mice were euthanized via isoflurane overdose and promptly exsanguinated by cardiocentesis. For transfusions using nonbiotinylated blood, naïve donor mice were euthanized. Whole blood was collected into sterile tubes in a 1:7 mixture with anticoagulant citrate phosphate dextrose adenine solution. The diluted anticoagulated blood (150 µL per mouse) then was administered to the recipient mice via retroorbital injection under anesthesia. Body weights for each recipient were recorded, and the total transfusion volume administered per mouse was no greater than 7.5% of the circulating blood volume, to limit suppression of normal erythropoiesis.

Experimental design.

The first group of mice served as controls for the determination of RBC survival in the C57BL/6 wildtype strain after direct injection of the biotinylating reagent. This experiment also was performed prior to proceeding with the principal aims of the study, to determine whether the RBC survival of male C57BL/6 mice significantly differed from that of female C57BL/6 mice. Each mouse received 1 mg of the biotinylating reagent, and blood samples were collected for flow cytometry on days 0, 1, 4, 8, 11, 15, 18, and 25 (until no more than 50% of the biotin-labeled RBC could be detected; Figure 1 C).

Figure 1.

Representative flow cytometric plots of biotinylated RBC. (A) Representative plot of nonbiotinylated, wildtype C57BL/6 RBC; 100% of RBC appear in the lower left (LL) quadrant. (B) At 2 d after biotinylation, blood samples collected from wildtype C57BL/6 mice are labeled with streptavidin–phycoerythrin (y-axis). The population of labeled RBC (>95%) appears in the upper left (UL) quadrant. Unlabeled RBC, including new cells emerging from the bone marrow, populate the LL quadrant. (C) At 30 d after biotinylation, 38.5% of biotin-labeled wildtype C57BL/6 RBC remain in circulation, visible in the upper left (UL) quadrant. At this time point, the population of unlabeled RBC in the LL quadrant represents young RBC, which were released from the bone marrow after the administration of biotinylating reagent.

Once the first experiment was completed, mice were assigned to groups (n = 20; 10 male and 10 female per group) to determine RBC survival in UBC–GFP and C57BL/6 wildtype mice after transfusion of biotinylated RBC. Blood samples for flow cytometry were collected every 2 to 5 d after transfusion until 50% or less of the day 0 percentage of biotinylated RBC remained in the recipient's circulation. Each group was divided into 2 cohorts, one in which RBC were transfused from biotinylated mice into recipients of the same strain as the donor, and the other into recipients of the opposite strain.

On day 90, all mice that had been transfused with biotinylated RBC received a second transfusion of biotinylated RBC. Blood again was collected from recipients at 2- to 5-d intervals for flow cytometry as for the first transfusion. Mice were euthanized on day 140, and serum was collected to screen for the presence of antibiotin antibodies (n = 40).

For the final experiment, C57BL/6 and UBC–GFP recipients (n = 10 per strain; 5 male and 5 female) were transfused with nonbiotinylated RBC from donor mice of the opposite strain. Blood samples for flow cytometry were collected every 2 to 5 d after transfusion until 50% or less of the initial population of transfused RBC remained in the recipient's circulation.

Blood sampling, streptavidin–phycoerythrin processing, and flow cytometry.

Transfusion recipients were sampled every 2 to 5 d through the collection of 3 to 6 µL of blood from a tail vein. Blood was collected into microfuge tubes containing 250 µL of sterile PBS and briefly vortexed. To detect biotinylated RBC from nonbiotinylated RBC on flow cytometry, approximately 10⁶ RBC (50 µL of the diluted sample) was added to 0.25 µg of streptavidin–phycoerythrin (1:100 of 0.5 mg/mL; BD Pharmingen). Samples were incubated in the dark for 5 min, washed with 400 µL PBS, and centrifuged at 1000 × g for 4 min. The RBC pellet was resuspended in 200 µL PBS, and sample analysis was conducted on an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA), with GFP-positive RBC detected on the first fluorescence channel (Figure 2), and streptavidin–phycoerythrin-positive RBC detected on the second fluorescence channel. Events were counted by using the Accuri software (BD Biosciences) and converted into percentages for each sample population.

Figure 2.

Representative flow cytometric plots of UBC-GFP and C57BL/6 RBC. (A) Representative plot of UBC-GFP mouse RBC. 98.4% of the RBC appear in the lower right (LR) quadrant with almost no RBC in the LL quadrant. (B) Representative plot of wildtype C57BL/6 mouse RBC; 100% of the RBC appear in the lower left (LL) quadrant. (C) Flow cytometric plot of RBC from a wildtype C57BL/6 mouse that had been transfused with RBC from a UBC–GFP donor 2 d earlier. The wildtype RBC are represented in the lower left (LL) quadrant, whereas the UBC–GFP RBC are clearly enumerated in the lower right (LR) quadrant.

Biotin antibody assay.

Mouse plasma samples (100 µL) were incubated with biotinylated RBC of UBC–GFP and C57BL/6 mice (10⁵ cells in 50 µL) for 5 to 10 min at room temperature. Samples were then washed with 500 µL PBS and centrifuged at 1000 × g for 5 min; 1 µL of secondary antibody (1:100 dilution; goat antimouse IgG polyclonal antibody, Abcam, Cambridge, MA) was added and incubated for 10 min. PBS (500 µL) was used to wash away excess secondary antibody prior to analysis. The detectable limit for antibiotin antibody was determined to be 0.1 µg/µL during optimization using monoclonal antibiotin antibodies produced in mice (Sigma, St Louis, MO).

Statistics.

Statistical analyses and linear regression were performed and graphed by using Prism 6 (GraphPad Software, La Jolla, CA). Statistical significance was defined as a P value of less than 0.05.

Results

Sex-associated difference in murine erythrocyte survival.

For male and female C57BL/6 mice, the percentage survival of biotinylated RBC over time decreased linearly at a rate of 2.3% ± 0.1% (female mice) and 2.23% ± 0.1% (male mice) daily (Figure 3 A). The rate at which biotinylated RBC were removed from circulation did not differ between male and female mice (P = 0.39); the observed half-life for the RBC lifespan 22.9 ± 1.2 d in male mice and 22.4 ± 0.9 d in female mice.

Figure 3.

RBC survival is not affected by sex or method of detection. (A) Male and female C57BL/6 mice were injected with 1 mg of biotinylating reagent; biotinylated RBC were detected by flow cytometry for 24 d after administration. No difference in RBC survival was detected between sexes (P = 0.39). (B) Biotinylated RBC from C57BL/6 and UBC–GFP mice were transfused into recipient mice of the same strain, and transfused RBC were tracked by flow cytometry for 24 d. There was no significant difference (N.S.; P = 0.21) in RBC survival between C57BL/6 and UBC–GFP mice. (C) Biotinylated RBC from C57BL/6 and UBC–GFP mice were transfused into recipients of the opposite strain. There was no significant difference (P = 0.26) between the RBC survival of C57BL/6 and UBC–GFP mice. (D) Unmodified RBC from C57BL/6 and UBC–GFP RBC were transfused into recipient mice of the opposite strain, and transfused RBC were tracked by flow cytometry. There was no significant difference between the RBC survival of C57BL/6 and UBC–GFP mice (P = 0.15).

RBC lifespans determined by transfusing biotinylated RBC.

The observed half-life for RBC lifespan in C57BL/6 mice, as determined through transfusion of biotinylated RBC from donor C57BL/6 mice, was 20.5 ± 2.1 d (–2.4% ± 0.2% daily; Figure 3 B). In comparison, the observed half-life for RBC lifespan in UBC–GFP mice, as determined through transfusion of biotinylated RBC from donor UBC–GFP mice, was 22.7 ± 2.1 d (–2.2 ± 0.1% daily; Figure 3 B). The rate at which biotinylated RBC were cleared from circulation did not differ between strains (P = 0.21). In addition, the half-life for RBC lifespan was similar regardless of whether it was determined by using direct biotinylation of mice or transfusion of biotinylated RBC (Figure 3 A).

Similarly, the rate of RBC clearance did not differ when biotinylated RBC were transfused into mice of the opposite strain (that is, biotinylated UBC–GFP RBC into C57BL/6 mice and vice versa; Figure 3 C). The percentage survival of transfused, biotinylated RBC declined at a linear rate in both groups at similar rates (P = 0.26; biotinylated C57BL/6 RBC into UBC–GFP mice: 2.4% ± 0.2%; T_1/2 = 20.2 ± 2.1 d; biotinylated UBC-GFP RBC into C57BL/6: 2.1% ± 0.2%; T_1/2 = 24.5 ± 3.3 d).

RBC survival in the absence of biotinylation.

The innate fluorescence of UBC-GFP RBC allowed us to determine RBC survival without biotinylating RBC (for example, flow cytometry differentiated UBC–GFP RBC transfused into C57BL/6 mice; Figure 2 C). The percentage survival of UBC–GFP RBC transfused into C57BL/6 mice declined at a linear rate (2.1% ± 0.2%; T_1/2 = 25.9 ± 3.3 d; Figure 3 D), as did the percentage RBC survival of C57BL/6 RBC transfused into UBC–GFP mice (2.3% ± 0.1%; T_1/2 = 20.4 ± 1.6 d; P = 0.15 for comparison between groups). The lifespan of nonbiotinylated RBC transfused into mice of the opposite strain did not differ significantly from that determined by using the biotinylating reagent in the earlier experiments.

RBC lifespan after repeat transfusion with biotinylated RBC.

To determine whether mice develop antibiotin antibodies, thus hampering the determination of RBC survival after repeated transfusions, we transfused a second dose of biotinylated RBC 90 d after the first transfusion. Biotinylated RBC administered in the first transfusion were cleared at a rate of 2.2% ± 0.1% daily (T_1/2 = 22.0 ± 1.2 d; Figure 4 A). This clearance rate did not differ significantly (P = 0.98) from that after the second transfusion (2.3% ± 0.1% daily; T_1/2 = 23.4 ± 1.4 d). Furthermore, antibodies to biotin were not detectable (at a limit of detection of 0.1 µg/µL), in recipient mice at 50 d after the second transfusion of biotinylated RBC (Figure 4 B).

Figure 4.

Subsequent transfusions of biotinylated RBC are unaffected by prior exposure. (A) Percentage survival of biotinylated RBC transfused 90 d after a prior exposure. The survival of biotinylated RBC transfused a second time did not significantly differ (P = 0.12) from that of the first exposure. (B) Plasma samples from mice transfused with biotinylated RBC were collected 50 d after the second exposure. The plasma was incubated with biotinylated RBC in vitro, and presence of antibiotin antibodies was detected by using a FITC-labeled antimouse IgG secondary antibody. Representative histograms of plasma samples are depicted along with a positive control sample, which was spiked with 1 µg/µL of antibiotin antibodies (+), and a negative saline control (–). No antibodies were detected from the 40 samples collected (indicated as different colors). N.S., no significant difference.

Discussion

Historically, radioisotope labeling by ⁵¹Cr has been considered the standard for determination of RBC survival and posttransfusion recovery in humans; however, several studies have shown excellent correlation with the biotin-labeling approach.^22,24,33,34 In addition, only minute blood sample volumes are required to run flow-cytometry analysis,⁸ making a biotin-labeling approach ideal for small laboratory animal species.^13,14 Therefore, we chose biotinylation as our reference method by which to compare RBC survival obtained by using UBC–GFP and wildtype C57BL/6 mice. In addition, we chose the C57BL/6 strain because it is one of the most widely used inbred strains for biomedical research.

The use of biotin labeling in RBC survival studies is well established; however, doses and methods for administration of biotinylating reagents vary widely in the literature, even in regard to the same species and strain of mouse.^10,18,30,35 Reported normal RBC lifespans among C57BL/6 mice range from 33 to 60 d (T_1/2 = 16.5 to 30 d).^1,17,18 Our study results fell within the average of this range, at an overall RBC half-life of 22.6 ± 0.7 d (lifespan of 45.2 d). Some of the variability observed in the literature may be due, in part, to subtle differences in labeling and sample processing techniques, which might unintentionally damage cells.⁵ Therefore, we developed a method for determining RBC survival that does not require labeling nor processing of RBC prior to detection by flow cytometry. We did not observe a significant difference in the RBC survival determined between biotinylation of RBC and using unmodified UBC-GFP RBC.

In addition, we did not note a difference in the RBC survival of C57BL/6 mice between sexes. Other RBC indices demonstrate slight differences between male and female mice, with female mice showing minimally higher RBC counts, Hct, and Hgb.^19,28 In other studies, female mice had a more robust erythropoietic response to chronic blood collection³¹ and more rapid hematopoietic stem-cell division in the bone marrow than did male mice.²⁹

One of the principal aims of our current study was to determine the circulating half-life of RBC in UBC–GFP mice, by using biotinylation and an unmodified RBC approach. We found no significant difference in the rate of RBC clearance from the circulation between C57BL/6 mice and UBC–GFP mice, regardless of the method of determination. This result suggests that GFP expression does not affect RBC senescence in this mouse strain. Furthermore, performing these cross-transfusion experiments enabled a direct, side-by-side comparison between the biotinylation and the GFP-based methods for determining RBC survival. As shown in Figure 3 C and D, there was no significant difference between the results.

Another aim of our study was to test whether administration of biotinylated RBC results in antibiotin antibody formation, potentially affecting subsequent RBC survival determinations. A small percentage of human subjects developed antibiotin antibodies after the transfusion of autologous biotinylated RBC.⁴ In addition, antibiotin antibodies have been found in serum samples from clinically normal humans.^2,6 The clinical relevance of these antibodies are unknown, but potential concerns are addressed in at least one study.⁴ To date, animal studies have not demonstrated similar antibiotin antibodies in plasma.³ We did not observe antibiotin antibodies in circulation 50 d after administering a dose of biotinylated RBC. Furthermore, there was no difference in the RBC survival results in subsequent RBC survival testing in mice that had been exposed to biotinylated RBC 90 d prior. However, we cannot rule out the presence nor the effect of these antibodies at other time points after primary exposure to biotinylated RBC.

In summary, we found no influence of sex on RBC survival in C57BL/6 mice. The RBC survival of UBC–GFP mice, which are on a C57BL/6 background, is equivalent to that of wildtype C57BL/6 mice. In addition, we optimized a method that does not require any labeling or processing of samples prior to detection of RBC by flow cytometry; this refinement has positive implications for studies characterizing new mouse models. By using UBC–GFP mice as recipients, RBC survival can be determined by transfusing them with nonGFP RBC from different strains on the C57BL/6 background (major histocompatibility complex haplotype H-2^b). As shown in Figure 2, populations of GFP and nonGFP RBC are easily tracked by using flow cytometry, without the need for labeling reagents or associated sample processing. Furthermore, we did not observe detectable levels of antibiotin antibodies after repeated transfusions of biotinylated RBC in mice, and there was no evidence of increased destruction of transfused RBC on repeated exposure to biotinylated RBC. Finally, all mice appeared clinically healthy throughout the posttransfusion monitoring period. Therefore, the described methods provide for a safe and effective way to determine mouse RBC survival.