Red Cell Volume Can Be Accurately Determined in Sheep Using a Nonradioactive Biotin Label

With the consensus among the scientific community in the late 1960’s that the use of radioactive materials was ethically untenable in research studies of fetuses, neonates, and pregnant women, the rate of progress in understanding red blood cell (RBC) kinetics and RBC volume in the perinatal period has nearly come to a halt (1). Yet, the need for understanding has continued to accelerate. Advances in obstetrics and in fetal medicine now permit intrauterine fetal blood sampling, transfusion, and other therapies to be delivered directly into the fetal circulatory system. Advances in neonatal medicine during this period include neonatal therapies that have resulted in improved survival and outcomes of extremely low birth weight (<1 kg) and other critically ill infants (2). While substantial evidence supports the use of surfactant, improved ventilator support, inhaled nitric oxide, and extracorporeal membrane oxygenation, other treatments such as erythropoietin and autologous placental blood for transfusion have weaker supporting bases of evidence and are being actively investigated. Reliable methods for measuring red blood cell volume (RCV) and survival in the pregnant woman, fetus, and infant are needed to further our understanding of the physiology and pathophysiology of a variety of perinatal conditions and their responses to treatment. Concerns about radiation effects have motivated the development of a practical method that does not expose infants, children, and pregnant women to radioactivity. RCV methods have been published that use stable isotopes followed by neutron activation (3) or X-ray fluorescence (4), but these methods require highly specialized, expensive equipment (3, 4), are not sufficiently sensitive for the small volumes of blood available from neonates (4), or both. As a result, neither of these methods has gained widespread use. The biotin RBC labeling method using either ex vivo (125I)streptavidin quantitation or flow cytometry enumeration is sufficiently sensitive (5-7) for use in determining RCV and RBC survival in neonates and adults without exposure to radiation. In addition, biotinylation reagents are commercially available (5, 7), and the equipment required is available in most medical center clinical laboratories (5, 7). Technician time required for analysis is modest. Because ovine fetal and newborn cardiovascular, pulmonary, and hematologic development resembles human physiology, the sheep model has been commonly used for preclinical studies. In particular, ovine erythropoiesis in the fetus, neonate, and adult is similar to the human; accordingly, regulation of erythropoiesis and RBC kinetics in the ovine model are currently areas of active study (8-11). An accurate, validated, practical method for determining RCV in the sheep would be useful for investigating neonatal anemia and cardiovascular problems. We previously demonstrated that the permanent RBC label (14C)cyanate is a valid alternative to 51Cr for measuring RCV in sheep (12). However, radioactive contamination presents a substantial disposal cost and (14C)cyanate is no longer commercially available. 51Cr is commercially available but is not suitable for RBC survival studies in sheep because Cr elutes from sheep RBCs over a few days. 51Cr also presents substantial disposal costs associated with the radioactive contamination of the animal and its excretion products. Thus, the objective of this study was to validate the use of the biotin labeling of RBCs and quantitation with both (125I)streptavidin detection and flow cytometry enumeration as alternative techniques for measuring RCV in sheep.