Avian RBCs have a short life span and high metabolic rate, which makes it challenging to maintain a viable supply of whole blood for treatment of birds with severe anemia.1–3 Hemoglobin-based oxygen carriers are not readily available to serve as blood substitutes in birds when homologous blood donors are unavailable. However, cryopreservation may allow for long-term storage of avian RBCs, thereby providing a useful blood supply when other blood sources are lacking.
Effects of storage media on RBCs from freshly collected blood of birds have been investigated. In 1 study,1 outcomes were evaluated after storage of avian RBCs in 3 commonly used media (0.9% citrate solution, acid-citrate-dextrose solution, and citrate-phosphate-dextrose solution with adenine). In all preparations, potassium concentrations increased substantially (> 30 mEq/L) after storage for 7 days, and stored blood was deemed unsafe for transfusion. To the authors’ knowledge, no other investigations of the effects of storage media on avian RBCs have been reported.
Outcomes of RBC transfusion have been investigated in pet bird species. In cockatiels (Nymphicus hollandicus), transfusions with RBCs harvested from the blood of autologous and homologous donors resulted in longer RBC survival times (10.5 to 16.8 days) than did transfusions with blood from heterologous donors (0.1 to 2.6 days). Results were different in conures of the genus Aratinga, in which RBC transfusions from autologous and homologous donors resulted in a half-life for RBCs of 9.9 and 8.5 days, respectively, and those from heterologous donors had a half-life of 4.5 days.3 Differences between RBC half-lives for transfusions with blood from autologous and homologous donors were not significant in that study.3 Consequently, homologous donors have been recommended as a source for blood transfusions in pet bird species,3 but heterologous donors may still be an option when homologous donors are not available.
Cryopreservation of RBCs allows for long-term (months to years) storage,4 providing a source of RBCs when other blood products are unavailable. Cryopreservation of cells typically involves use of glycerol or DMSO as a cryoprotectant; however, these media are toxic and must be removed from RBC samples prior to transfusion. The process for removal of cryoprotectants is time consuming, and addition and removal of glycerol or DMSO can cause osmotic stress to cells.4 Although DMSO has been used to successfully cryopreserve RBCs from hens for use as reagents in virus titration studies, glycerol is considered unfit for use in cryopreservation because the cell recovery rate after thawing and washing is poor.5 Effects of extracellular additives that do not penetrate RBCs have also been investigated. Hydroxyethyl starch solution has been used for cryopreservation of human and canine RBCs,6,7 with the advantage that HES is nontoxic and RBCs preserved with it can be administered to patients without the need for cell washing prior to transfusion.
A study8 revealed that human RBCs stored in HES solution may need to be washed prior to transfusion to reduce the amount of free hemoglobin in the transfused product. However, a later study9 revealed that autologous reinfusion of human RBCs that had been cryopreserved in HES solution led to no adverse reactions, regardless of whether cells were washed after thawing. In another study,10 some humans developed mild leukocytosis and a moderate increase in serum bilirubin concentration after transfusion with RBCs stored in HES solution, but these effects were no longer evident 20 hours after transfusion.
Elimination of HES from the bloodstream reportedly follows first-order kinetics in dogs and humans.11 Intravenous infusion of HES solution has been successfully used in the resuscitation of birds with shock caused by severe blood loss.12 Interestingly, oxyglobin was more effective for resuscitation than was HES or crystalloid solutions in that study.12 It follows that RBCs cryopreserved with HES solution may also be more effective than HES or crystalloid solution alone for resuscitation of birds with severe blood loss.
Concentration of an HES solution can affect degree of RBC recovery after cryopreservation. In dogs, a 12.5% (wt/vol) concentration was identified as yielding a greater proportion of viable RBCs after cryopreservation than did other concentrations of HES solution.7 In humans, transfusion with washed or unwashed RBCs cryopreserved in 11.5% HES solution reportedly yields results similar to those of transfusion with RBCs stored in solution containing phosphate, adenine, glucose, guanosine, saline solution, and mannitol.8 Another study13 revealed a cell recovery rate of 97% and saline stability > 80% when human RBCs were cryopreserved in 14% HES solution. An in vivo study14 in dogs revealed no difference in 24-hour and long-term survival rates between radiolabeled, autologous RBCs cryopreserved with HES solution and freshly donated autologous RBCs, with > 95% of HES-cryopreserved cells surviving 24 hours after transfusion.
Integrity of RBCs can be assessed on the basis of several criteria, including degree of stability in isotonic saline (0.9% NaCl) solution, plasma stability, percentage of cells recovered, mean corpuscular volume, extracellular electrolyte concentrations, hemoglobin content, intracellular and extracellular pH, intracellular lactate concentration, density gradients, osmotic fragility, and cell morphology as assessed via scanning electron microscopy.7,15 In general, percentages of RBCs recovered after freezing should be > 80% after any washing or cryoprotectant-removal process, and degree of RBC stability in saline solution during a 30-minute period should be > 85%.16
The reagents 7-AAD and annexin V are used to identify cellular death and apoptosis, respectively,17,18 and are therefore also used to assess cell viability following cryopreservation.19 Markers of apoptosis are used to detect translocation of phosphatidylserine to the outer plasma membrane, which occurs in the early stages of apoptosis and necrosis. Apoptosis and erythrocyte senescence (eryptosis) result in exposure of phosphatidylserine in the outer membrane of cells.20 Although 7-AAD and annexin V have not specifically been used to measure viability of avian erythrocytes, they have been used together to characterize the presence and stage of apoptosis in suspensions of avian lymphocytes.21 The objective of the study reported here was to compare the effectiveness of glycerol, 10% DMSO, and HES solutions for the cryopreservation of avian RBCs. Our hypothesis was that effectiveness would differ significantly among media.
Supported by the Morris Animal Foundation.
Presented in abstract form at the 2014 Annual Conference & Expo of the Association of Avian Veterinarians, New Orleans, August 2014, and at the 46th Annual Conference of the Association of American Zoological Veterinarians, Orlando, Fla, October 2014.
The authors thank Airiel Davis for technical assistance.
Fisherbrand Easy Reader polypropylene centrifuge tubes (flat top closure, sterile, in rack), 15 mL, Fisher Scientific, Pittsburgh, Pa.
Sigma Chemical Co, St Louis, Mo.
HyClone defined fetal bovine serum (SH30070.03), 500 mL, GE Healthcare Life Sciences, Logan, Utah.
MEM alpha Eagle medium, BioWhittaker Cell Culture Media, Lonza, Allendale, NJ.
KryoHAES (molecular weight, 200,000 Da; degree of substitution, 0.5; concentration, 23% wt/wt), Fesenius, Bad Homburg, Germany.
ELx808 Absorbance Reader, BioTek Winooski, Vt.
BD Biosciences, San Jose, Calif.
Biotium Inc, Hayward, Calif.
10× Annexin V binding buffer, BD Biosciences, San Diego, Calif.
FEI Quanta 200 FESEM MK II, FEI, Hillsboro, Ore.
SAS, version 9.3, SAS Institute Inc, Cary, NC.
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