Post-transfusion survival of biotin-labeled allogeneic RBCs in adult horses.

BACKGROUND: Post-transfusion survival of allogeneic RBCs has been reported to be much shorter in horses than in other species. We hypothesized that post-transfusion survival of biotinylated allogeneic equine RBCs would be greater than the survival previously reported from studies using radioactive RBC-labeling techniques. OBJECTIVE: The study objective was to determine post-transfusion survival of N-hydroxysuccinimide (NHS)-biotin-labeled allogeneic equine RBCs transfused into adult horses. METHODS: Horses were adults and included 5 donors and 5 recipients. All horses were blood-typed, and donors were paired with recipients based upon blood type and crossmatch results. Donor blood was collected in a volume of 4 L into citrate phosphate dextrose adenine-1 and stored for 24 hours, labeled with NHS-biotin, and re-infused into recipients. Post-transfusion blood samples were collected at 15 minutes and at 1, 2, 3, 5, 7, 14, 21, 28, and 35 days. Biotin-labeled RBCs were detected by flow cytometry using streptavidin-phycoerythrin. Post-transfusion survival at 24 hours, lifespan, and half-life of biotinylated RBCs were determined. RESULTS: Mean ± SD survival of biotinylated RBCs at 24 hours post-transfusion was 95 ± 24%; the mean lifespan of transfused allogeneic RBCs was 39 days based on calculation of a linear regression survival curve, and mean post-transfusion RBC half-life was 20 days. CONCLUSIONS: Post-transfusion survival of 24-hour stored equine allogeneic RBCs was greater than previously reported but less than that observed for other companion animal species. Mechanisms for the relatively short post-transfusion lifespan of allogeneic equine RBCs remain unknown and warrant further study.

Processing of equine bone marrow using the automated MarrowXpress System: RBC depletion, volume reduction, and mononuclear cell recovery.

BACKGROUND: The therapeutic use of bone marrow-derived mononuclear cells (MNCs) and mesenchymal stem cells for the treatment of soft tissue and orthopedic injuries in equine patients is expanding. After collection, bone marrow must be reduced in volume and depleted of RBCs for immediate therapeutic use or to prepare cells for culture or cryopreservation and storage. The MarrowXpress (MXP) System is an automated, closed, sterile system designed to process human bone marrow samples. OBJECTIVES: The purpose of this study was to evaluate the capacity of the MXP System to process equine bone marrow to reduce volume, deplete RBCs, and enhance recovery of MNCs. METHODS: Bone marrow was collected from 47 horses into 2 60-mL syringes containing heparin and processed using the MXP System. HCT, total nucleated cell (TNC) count, and MNC count were obtained for each sample before and after processing using an Advia 120 hematology analyzer. Volume reduction, RBC depletion, and recovery of TNCs and MNCs were calculated. RESULTS: For equine bone marrow samples, mean values were 73.2% for RBC depletion and 78.0% for volume reduction. TNC count before processing was 2.5 ± 1.2 × 10(7) and after processing was significantly higher at 7.8 ± 3.3 × 10(7) (P < .0001), with a recovery of 68.5 ± 24.5% (mean ± SD). MNC count before processing was 1.1 ± 0.9 × 10(7) and after processing was significantly higher at 3.8 ± 1.9 × 10(7) (P < .0001), with a recovery 73.0 ± 31.5%. CONCLUSIONS: The MXP System can reliably reduce volume and deplete RBCs from aspirates of equine bone marrow aspirates. MNCs can be recovered in a reproducible and sterile manner. Further studies evaluating the effects of the MXP System on cell viability, identification of mesenchymal stem cells (MSCs), and the efficacy of MSC expansion are warranted.

Clinicopathologic findings following intra-articular injection of autologous and allogeneic placentally derived equine mesenchymal stem cells in horses.

BACKGROUND AIMS: The development of an allogeneic mesenchymal stem cell (MSC) product to treat equine disorders would be useful; however, there are limited in vivo safety data for horses. We hypothesized that the injection of self (autologous) and non-self (related allogeneic or allogeneic) MSC would not elicit significant alterations in physical examination, gait or synovial fluid parameters when injected into the joints of healthy horses. METHODS: Sixteen healthy horses were used in this study. Group 1 consisted of foals (n = 6), group 2 consisted of their dams (n = 5) and group 3 consisted of half-siblings (n = 5) to group 1 foals. Prior to injection, MSC were phenotyped. Placentally derived MSC were injected into contralateral joints and MSC diluent was injected into a separate joint (control). An examination, including lameness evaluation and synovial fluid analysis, was performed at 0, 24, 48 and 72 h post-injection. RESULTS: MSC were major histocompatibility complex (MHC) I positive, MHC II negative and CD86 negative. Injection of allogeneic MSC did not elicit a systemic response. Local responses such as joint swelling or lameness were minimal and variable. Intra-articular MSC injection elicited marked inflammation within the synovial fluid (as measured by nucleated cell count, neutrophil number and total protein concentration). However, there were no significant differences between the degree and type of inflammation elicited by self and non-self-MSC. CONCLUSIONS: The healthy equine joint responds similarly to a single intra-articular injection of autologous and allogeneic MSC. This pre-clinical safety study is an important first step in the development of equine allogeneic stem cell therapies.

Use of an in vitro biotinylation technique for determination of posttransfusion survival of fresh and stored autologous red blood cells in Thoroughbreds.

OBJECTIVE: To evaluate N-hydroxysuccinimide (NHS)-biotin labeling of equine RBCs and determine posttransfusion survival of autologous equine RBCs stored in citrate phosphate dextrose adenine-1 (CPDA-1) for 0, 1, 14, and 28 days. ANIMALS: 13 healthy adult Thoroughbreds. PROCEDURES: Serial dilutions of biotin and streptavidin-phycoerythrin (PE) were evaluated in vitro in blood collected from 3 horses. One horse was used to determine RBC distribution and recovery. Twelve horses were allocated to 4 groups for in vivo experiments in which blood was collected into CPDA-1. Blood was labeled with biotin and reinfused or stored at 4 degrees C for 1, 14, or 28 days prior to labeling with NHS-biotin and reinfusion. Posttransfusion blood samples were collected 15 minutes and 1, 2, 3, 5, 7, 14, 21, 28, and 35 days after reinfusion. Biotin-labeled RBCs were detected via flow cytometry by use of streptavidin-PE. Posttransfusion lifespan of RBCs and RBC half-life were determined. RESULTS: Optimal biotin concentration was 0.04 pg of biotin/RBC, and the optimal streptavidin-PE ratio was 1.2 microg of streptavidin-PE/1 x 10(6) RBCs. Posttransfusion lifespan of autologous RBCs was 99, 89, 66, and 59 days after storage for 0, 1, 14, and 28 days, respectively. Storage did not result in significant alterations in RBC lifespan. Mean posttransfusion RBC half-life was 50, 45, 33, and 29 days for 0, 1, 14, and 28 days of storage, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Biotin can be used to label equine RBCs for RBC survival studies. Posttransfusion survival of equine autologous RBCs was greater than previously reported.