Human Serum Extends Survival of Transfused Human Red Blood Cells in Immunodeficient Mice

Introduction: Humanized mouse models are tools for preclinical research. The survival of human blood cells in the circulation of immunodeficient mice is poor, leading to reduce overall engraftment and limiting research applications. Previous research indicated that human AB serum (HABS) could enhance human cell survival in immunodeficient mice. We sought to confirm these data and to determine what component of HABS is responsible for promoting human cell survival.

Methods: Three cohorts of 8-10-week-old NSG mice were used to study human red blood cell (hRBC) survival. hRBCs were collected from healthy volunteers and transfused into mice. Blood samples were analyzed for hematocrit and hRBCs count. The first cohort compared hRBCs survival with human, mouse, and bovine serum (FBS) administered intraperitoneally (IP). The second cohort tested the effect of HABS titration and administration routes (IV and IP) on hRBCs survival. The third cohort identified the role of human serum fractions (HABS, Cohn fraction II/III, purified IgG, and IgG-depleted serum) in hRBCs survival. Mice were bled at different times post-transfusion, stained, and analyzed via flow cytometry. Statistical analysis was conducted using Prism software with significant results at P ≤ 0.05.

Results: Transfusion of hRBCs in mice treated with HABS resulted in significantly higher longevity than mice without any treatment over 3 hours after injection. Total area under the curve (AUC) of hRBCs transfused with PBS was 8169.0±416.5 and with HABS was 14192.0±1675. Subsequently, NSG mice were treated with NSG mouse serum, HABS, or BALBc mouse serum to determine whether any non-NSG sera would prolong hRBCs survival. Only NSG mice treated with HABS had significantly higher hRBCs frequencies over time (AUC: 13293.0±1777), whereas mice treated with autologous NSG serum had an AUC of 7636.0±1553.0 and BALBc serum resulted in a mild clearance effect (AUC: 3271.0±315.1). hRBCs delivered with different FBS sources had no effect when compared against HABS. Moreover, we were able to detect viable hRBCs up to 12 hours post transfusion with a single dose of HABS. Additionally, we tested if a second (5 hours after transfusion) or third (30 hours after transfusion) HABS dose would further improve this outcome, but there was no difference as shown my AUC comparisons.

To determine the optimal volume of HABS required to prolong hRBCs survival, NSG mice received 100 ml hRBCs resuspended in 200 - 500 ml of HABS via intravenous (IV) injection. A dosage-dependent positive correlation was observed: 300 and 400 ml HABS provided a significantly higher hRBCs frequencies than PBS over the course of 3 hours and with no difference in the effect as compared to IP injection. Furthermore, a comparison between administering HABS IV (15441±421.6), or by both IV and IP routes (15854±461.2) showed a greater effect than solely administering HABS via IP (12076±1445). Thus, suspending hRBCs in HABS prior IV administration increases their survival.

Further analysis of human serum components showed that HABS, human serum Cohn fraction II/III, and pure human IgG also improved hRBCs survival in NSG mice. Furthermore, titration of purified IgG showed a positive effect with concentrations starting at 5 mg (AUC: 74280±4033) when compared to PBS-only (AUC: 43070±3289).

Conclusion: Taken together this data strongly suggest HABS significantly improves survival of circulating hRBCs transfused in NSG mice as proposed before. Moreover, hRBCs frequencies can be monitored up to 12 hours post-transfusion just by adding HABS. Additionally, human IgG may be the active component of HABS that is responsible for this effect through, has yet unknown mechanisms.

No relevant conflicts of interest to declare.