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

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

Urshulaa Dholakia,1* Sheila Bandyopadhyay,2 Eldad A Hod,2 and Kevin A Prestia1,2

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).

Abbreviation: T1/2, half-life.

RBC lifespan and senescence are important parameters used transfusion of biotinylated RBC. Although it is generally accepted both clinically and in research studies of hereditary disorders of that biotinylation of RBC does not affect their function, antibodies to erythrocyte metabolism, transfusion medicine, and sepsis.8,21,27,32,35 biotin have been demonstrated in some human studies, posing the Labeling RBC with a biotinylating reagent is a common method question of whether repeated administration of biotin ester or bioti- used to determine their circulating lifespan. Other methods involve nylated RBC could interfere with subsequent results within the same using radioactive isotopes, such as 51Cr and 59Fe.7,20 Biotinylating subject.4,20 Repeat transfusions of biotinylated RBC to mice have not reagents are preferred for various research applications with hu- been described in the literature. One aim of this study was to deter- mans,8,23,24 and are used in a variety of animal models.1,25,33,34,37 Once mine whether exposure to biotinylated RBC induces an antibiotin biotin attaches to RBC surface proteins, streptavidin (a protein de- antibody response in mice. Furthermore, we tested whether the sur- rived from Streptomyces avidinii) that is labeled with a fluorescent vival of biotinylated RBC changed after repeat exposure. dye is used to form a strong and rapid complex with biotin, thereby Recently GFP-expressing RBC have been used to track the post- allowing for its detection through flow cytometry. Blood samples transfusion survival and recovery of stored RBC administered to analyzed sequentially over a period of weeks will show a linear nonGFP-expressing recipient mice.9,12,36 The C57BL/6-Tg(UBC– decline in biotin–streptavidin signal as labeled cells age and are GFP)30Scha/J mouse strain is characterized by GFP expression cleared from the circulation through the reticuloendothelial system. under the control of a human ubiquitin C promoter. All tissues The characteristics of an ideal label for performing RBC survival of these mice express GFP, including blood.26 GFP expression ap- studies include: 1) stable presence on or within the cell throughout pears to be consistent throughout life and does not otherwise alter its normal lifespan; 2) specificity for RBC; 3) inertness, such that the the normal structure, physiology, or function of RBC. In addi- cell does not become prone to accelerated destruction; 4) nonrecy- tion, GFP is unaffected by ambient light contamination or deg- cling (that is, the label does not reenter the circulation and bind to radation, drawbacks that are associated with fluorescent dyes.15 new cells after destruction of the labeled RBC); and 5) easy and ac- In addition, GFP allows for the separation of cell populations curate measurement by using available assays. Radioactive isotopes through flow cytometry.9,11 Many qualities of GFP suggest that it and other labels fulfill several of these criteria, but their limitations may serve as a useful surrogate marker in place of other labeling include elution from RBC as well as safety concerns.7,22 In contrast, techniques in mice. Therefore, we sought to evaluate the utility of biotin poses little to no risk of accumulation or toxicity. The sulfo- N- UBC–GFP transgenic mice as an alternative to labeling RBC with hydroxysuccinimide–biotin ester used for RBC tracking studies in biotin esters. Our aim for this work was to determine the survival humans and animals can be administered directly or through the of RBC in wild-type C57BL/6 mice and in the UBC–GFP strain

and to compare methods for determining RBC half-life (T1/2). Received: 13 Nov 2014. Revision requested: 12 Jan 2015. Accepted: 25 Jan 2015. 1Institute of Comparative Medicine, Columbia University, and 2Laboratory of Transfusion Biology, Department of Pathology and Cell Biology, College of Physicians and Surgeons, Materials and Methods Columbia University, New York, New York Animals. Male and female C57BL/6 mice (age, 6 to 8 wk) were *Corresponding author. Email: [email protected]. purchased from Charles River Laboratories (Wilmington, MA), 196 Determination of RBC survival in mice

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.

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. and male and female C57BL/6-Tg(UBC-GFP)30Schaa/J mice mouse adenoviruses, mouse parvoviruses, mouse hepatitis virus, (age, 6 to 8 wk) were acquired from Jackson Laboratories (stock Theiler murine encephalomyelitis virus, mouse rotavirus, Sendai no., 004353; Bar Harbor ME). Mice were housed in compatible virus, pneumonia virus of mice, mouse reovirus type 3, lympho- single-sex groups within static microisolator cages, provided cytic choriomeningitis virus, ectromelia virus, Hantaan virus, with free choice standard rodent chow diet (Purina Picolab 5058; Mycoplasma pulmonis, and endo- and ectoparasites). LabDiet, St Louis, MO), autoclaved tap water, and autoclaved Biotinylation and RBC transfusions. EZ-Link Sulfo-NHS Biotin corncob bedding, cotton nesting material, and environmental (Pierce, Rockford, IL) was prepared in the laboratory for intra- enrichment (Shepherd Shacks, Shepherd Specialty Papers, Wa- venous injection. The powder was dissolved in sterile PBS, vor- tertown, TN). Animal protocols were approved by the IACUC texed, and passed through a 0.22-µm filter and diluted to a final of Columbia University (New York, NY), and all rodent health concentration of 1 mg biotin per 300-µL injection. Mice received monitoring, husbandry, and experimental procedures were per- the biotin reagent solution intravenously by retroorbital injection formed in compliance with the recommendations of the Guide under anesthesia. for the Care and Use of Laboratory Animals.16 The animal facility is For transfusions of biotinylated RBC, 2 male donor mice AAALAC-accredited and maintained at an SPF status (free of of the selected strain received the biotinylating reagent as

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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). described earlier. At 2d after injection, mice were euthanized days 0, 1, 4, 8, 11, 15, 18, and 25 (until no more than 50% of the via isoflurane overdose and promptly exsanguinated by car- biotin-labeled RBC could be detected; Figure 1 C). diocentesis. For transfusions using nonbiotinylated blood, na- Once the first experiment was completed, mice were assigned ïve donor mice were euthanized. Whole blood was collected to groups (n = 20; 10 male and 10 female per group) to determine into sterile tubes in a 1:7 mixture with anticoagulant citrate RBC survival in UBC–GFP and C57BL/6 wildtype mice after phosphate dextrose adenine solution. The diluted anticoagu- transfusion of biotinylated RBC. Blood samples for flow cytom- lated blood (150 µL per mouse) then was administered to the etry were collected every 2 to 5 d after transfusion until 50% or recipient mice via retroorbital injection under anesthesia. Body less of the day 0 percentage of biotinylated RBC remained in the weights for each recipient were recorded, and the total transfu- recipient’s circulation. Each group was divided into 2 cohorts, one sion volume administered per mouse was no greater than 7.5% in which RBC were transfused from biotinylated mice into recipi- of the circulating blood volume, to limit suppression of normal ents of the same strain as the donor, and the other into recipients erythropoiesis. of the opposite strain. Experimental design. The first group of mice served as controls On day 90, all mice that had been transfused with biotinylated for the determination of RBC survival in the C57BL/6 wildtype RBC received a second transfusion of biotinylated RBC. Blood strain after direct injection of the biotinylating reagent. This ex- again was collected from recipients at 2- to 5-d intervals for flow periment also was performed prior to proceeding with the prin- cytometry as for the first transfusion. Mice were euthanized on cipal aims of the study, to determine whether the RBC survival day 140, and serum was collected to screen for the presence of of male C57BL/6 mice significantly differed from that of female antibiotin antibodies (n = 40). C57BL/6 mice. Each mouse received 1 mg of the biotinylating For the final experiment, C57BL/6 and UBC–GFP recipients reagent, and blood samples were collected for flow cytometry on (n = 10 per strain; 5 male and 5 female) were transfused with

198 Determination of RBC survival in mice

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.

Statistics. nonbiotinylated RBC from donor mice of the opposite strain. Statistical analyses and linear regression were performed Blood samples for flow cytometry were collected every 2 to 5 and graphed by using Prism 6 (GraphPad Software, La Jolla, CA). P d after transfusion until 50% or less of the initial population of Statistical significance was defined as a value of less than 0.05. transfused RBC remained in the recipient’s circulation. Blood sampling, streptavidin–phycoerythrin processing, and Results flow cytometry. Transfusion recipients were sampled every 2 Sex-associated difference in murine erythrocyte survival. For to 5 d through the collection of 3 to 6 µL of blood from a tail male and female C57BL/6 mice, the percentage survival of bioti- vein. Blood was collected into microfuge tubes containing 250 nylated RBC over time decreased linearly at a rate of 2.3% ± 0.1% µL of sterile PBS and briefly vortexed. To detect biotinylated (female mice) and 2.23% ± 0.1% (male mice) daily (Figure 3 A). RBC from nonbiotinylated RBC on flow cytometry, approxi- The rate at which biotinylated RBC were removed from circula- mately 106 RBC (50 µL of the diluted sample) was added to tion did not differ between male and female mice (P = 0.39); the 0.25 µg of streptavidin–phycoerythrin (1:100 of 0.5 mg/mL; observed half-life for the RBC lifespan 22.9 ± 1.2 d in male mice BD Pharmingen). Samples were incubated in the dark for 5 and 22.4 ± 0.9 d in female mice. min, washed with 400 µL PBS, and centrifuged at 1000 × g for RBC lifespans determined by transfusing biotinylated RBC. 4 min. The RBC pellet was resuspended in 200 µL PBS, and The observed half-life for RBC lifespan in C57BL/6 mice, as de- sample analysis was conducted on an Accuri C6 flow cytom- termined through transfusion of biotinylated RBC from donor eter (BD Biosciences, San Jose, CA), with GFP-positive RBC C57BL/6 mice, was 20.5 ± 2.1 d (–2.4% ± 0.2% daily; Figure 3 B). detected on the first fluorescence channel (Figure 2), and strep- In comparison, the observed half-life for RBC lifespan in UBC– tavidin–phycoerythrin-positive RBC detected on the second GFP mice, as determined through transfusion of biotinylated RBC fluorescence channel. Events were counted by using the Accuri from donor UBC–GFP mice, was 22.7 ± 2.1 d (–2.2 ± 0.1% daily; software (BD Biosciences) and converted into percentages for Figure 3 B). The rate at which biotinylated RBC were cleared from each sample population. circulation did not differ between strains (P = 0.21). In addition, Biotin antibody assay. Mouse plasma samples (100 µL) were the half-life for RBC lifespan was similar regardless of whether it incubated with biotinylated RBC of UBC–GFP and C57BL/6 mice was determined by using direct biotinylation of mice or transfu- (105 cells in 50 µL) for 5 to 10 min at room temperature. Samples sion of biotinylated RBC (Figure 3 A). were then washed with 500 µL PBS and centrifuged at 1000 × g for Similarly, the rate of RBC clearance did not differ when bio- 5 min; 1 µL of secondary antibody (1:100 dilution; goat antimouse tinylated RBC were transfused into mice of the opposite strain IgG polyclonal antibody, Abcam, Cambridge, MA) was added (that is, biotinylated UBC–GFP RBC into C57BL/6 mice and vice and incubated for 10 min. PBS (500 µL) was used to wash away versa; Figure 3 C). The percentage survival of transfused, bioti- excess secondary antibody prior to analysis. The detectable limit nylated RBC declined at a linear rate in both groups at similar for antibiotin antibody was determined to be 0.1 µg/µL during rates (P = 0.26; biotinylated C57BL/6 RBC into UBC–GFP mice: optimization using monoclonal antibiotin antibodies produced 2.4% ± 0.2%; T1/2 = 20.2 ± 2.1 d; biotinylated UBC-GFP RBC into in mice (Sigma, St Louis, MO). C57BL/6: 2.1% ± 0.2%; T1/2 = 24.5 ± 3.3 d). 199 Vol 65, No 3 Comparative Medicine June 2015

RBC survival in the absence of biotinylation. The innate fluo- nylation and an unmodified RBC approach. We found no signifi- rescence of UBC-GFP RBC allowed us to determine RBC survival cant difference in the rate of RBC clearance from the circulation without biotinylating RBC (for example, flow cytometry differ- between C57BL/6 mice and UBC–GFP mice, regardless of the entiated UBC–GFP RBC transfused into C57BL/6 mice; Figure method of determination. This result suggests that GFP expression 2 C). The percentage survival of UBC–GFP RBC transfused into does not affect RBC senescence in this mouse strain. Furthermore,

C57BL/6 mice declined at a linear rate (2.1% ± 0.2%; T1/2 = 25.9 ± performing these cross-transfusion experiments enabled a direct, 3.3 d; Figure 3 D), as did the percentage RBC survival of C57BL/6 side-by-side comparison between the biotinylation and the GFP-

RBC transfused into UBC–GFP mice (2.3% ± 0.1%; T1/2 = 20.4 ± based methods for determining RBC survival. As shown in Figure 1.6 d; P = 0.15 for comparison between groups). The lifespan of 3 C and D, there was no significant difference between the results. nonbiotinylated RBC transfused into mice of the opposite strain Another aim of our study was to test whether administration did not differ significantly from that determined by using the of biotinylated RBC results in antibiotin antibody formation, po- biotinylating reagent in the earlier experiments. tentially affecting subsequent RBC survival determinations. A RBC lifespan after repeat transfusion with biotinylated RBC. To small percentage of human subjects developed antibiotin anti- determine whether mice develop antibiotin antibodies, thus ham- bodies after the transfusion of autologous biotinylated RBC.4 In pering the determination of RBC survival after repeated transfu- addition, antibiotin antibodies have been found in serum samples sions, we transfused a second dose of biotinylated RBC 90 d after from clinically normal humans.2,6 The clinical relevance of these the first transfusion. Biotinylated RBC administered in the first antibodies are unknown, but potential concerns are addressed in 4 transfusion were cleared at a rate of 2.2% ± 0.1% daily (T1/2 = 22.0 at least one study. To date, animal studies have not demonstrated ± 1.2 d; Figure 4 A). This clearance rate did not differ significantly similar antibiotin antibodies in plasma.3 We did not observe an- (P = 0.98) from that after the second transfusion (2.3% ± 0.1% daily; tibiotin antibodies in circulation 50 d after administering a dose

T1/2 = 23.4 ± 1.4 d). Furthermore, antibodies to biotin were not de- of biotinylated RBC. Furthermore, there was no difference in the tectable (at a limit of detection of 0.1 µg/µL), in recipient mice at RBC survival results in subsequent RBC survival testing in mice 50 d after the second transfusion of biotinylated RBC (Figure 4 B). that had been exposed to biotinylated RBC 90 d prior. However, we cannot rule out the presence nor the effect of these antibodies Discussion at other time points after primary exposure to biotinylated RBC. In summary, we found no influence of sex on RBC survival Historically, radioisotope labeling by 51Cr has been considered in C57BL/6 mice. The RBC survival of UBC–GFP mice, which the standard for determination of RBC survival and posttransfu- are on a C57BL/6 background, is equivalent to that of wildtype sion recovery in humans; however, several studies have shown C57BL/6 mice. In addition, we optimized a method that does not excellent correlation with the biotin-labeling approach.22,24,33,34 In require any labeling or processing of samples prior to detection addition, only minute blood sample volumes are required to run of RBC by flow cytometry; this refinement has positive impli- flow-cytometry analysis,8 making a biotin-labeling approach ideal cations for studies characterizing new mouse models. By using for small laboratory animal species.13,14 Therefore, we chose bioti- UBC–GFP mice as recipients, RBC survival can be determined by nylation as our reference method by which to compare RBC sur- transfusing them with nonGFP RBC from different strains on the vival obtained by using UBC–GFP and wildtype C57BL/6 mice. C57BL/6 background (major histocompatibility complex haplo- In addition, we chose the C57BL/6 strain because it is one of the type H-2b). As shown in Figure 2, populations of GFP and non- most widely used inbred strains for biomedical research. GFP RBC are easily tracked by using flow cytometry, without the The use of biotin labeling in RBC survival studies is well estab- need for labeling reagents or associated sample processing. Fur- lished; however, doses and methods for administration of bio- thermore, we did not observe detectable levels of antibiotin an- tinylating reagents vary widely in the literature, even in regard tibodies after repeated transfusions of biotinylated RBC in mice, to the same species and strain of mouse.10,18,30,35 Reported normal and there was no evidence of increased destruction of transfused RBC lifespans among C57BL/6 mice range from 33 to 60 d (T = 1/2 RBC on repeated exposure to biotinylated RBC. Finally, all mice 16.5 to 30 d).1,17,18 Our study results fell within the average of this appeared clinically healthy throughout the posttransfusion moni- range, at an overall RBC half-life of 22.6 ± 0.7 d (lifespan of 45.2 toring period. Therefore, the described methods provide for a safe d). Some of the variability observed in the literature may be due, and effective way to determine mouse RBC survival. in part, to subtle differences in labeling and sample processing techniques, which might unintentionally damage cells.5 There- fore, we developed a method for determining RBC survival that Acknowledgments does not require labeling nor processing of RBC prior to detection We thank Drs Steven Spitalnik, and Brian Karolewski for their support by flow cytometry. We did not observe a significant difference in and encouragement. the RBC survival determined between biotinylation of RBC and using unmodified UBC-GFP RBC. References In addition, we did not note a difference in the RBC survival 1. Chan JY, Kwong M, Lo M, Emerson R, Kuypers FA. 2001. Reduced of C57BL/6 mice between sexes. Other RBC indices demonstrate oxidative-stress response in red blood cells from p45NFE2-deficient slight differences between male and female mice, with female mice. Blood 97:2151–2157. mice showing minimally higher RBC counts, Hct, and Hgb.19,28 2. Chen T, Hedman L, Mattila PS, Jartti L, Jartti T, Ruuskanen O, In other studies, female mice had a more robust erythropoietic Soderlund-Venermo M, Hedman K. 2012. Biotin IgM antibodies response to chronic blood collection31 and more rapid hematopoi- in human blood: a previously unknown factor eliciting false results 29 in biotinylation-based immunoassays. PLoS ONE 7:e42376. etic stem-cell division in the bone marrow than did male mice. 3. Christian JA, Rebar AH, Boon GD, Low PS. 1993. Senescence of canine One of the principal aims of our current study was to determine biotinylated erythrocytes: increased autologous immunoglobulin the circulating half-life of RBC in UBC–GFP mice, by using bioti-

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