Gary L. Lankford

Measurement of red cell survival using biotin-labeled red cells: validation against 51Cr-labeled red cells

BACKGROUND: Anemia is a serious problem in the fetus and preterm infant. To investigate the physiology and pathophysiology of anemia and to assess responses to blood transfusions and erythropoietin therapy, measurement of circulating red cell survival would be useful.

Measurement of circulating red cell volume using biotin‐labeled red cells: validation against 51Cr‐labeled red cells

BACKGROUND: Anemia is a serious problem in the fetus and preterm infant. To investigate the physiology and pathophysiology of anemia and to assess responses to blood transfusions or erythropoietin therapy, measurement of circulating red cell volume would be useful. Because the standard 51Cr method exposes the subject to radiation, a method of measuring circulating red cell volume without radiation exposure, sufficiently sensitive for use in fetuses and infants, was developed.

Antibodies provoked by the transfusion of biotin-labeled red cells

BACKGROUND: Biotin‐labeled (biotinylated) red cells (B‐RBCs) offer a technique by which to study RBC volume and circulating kinetics without in vivo radiation. The immunogenicity of B‐RBCs is undefined.

Hematocrit Correlates Well with Circulating Red Blood Cell Volume in Very Low Birth Weight Infants

Although circulating red blood cell (RBC) volume is a better measure of total body oxygen delivering capacity than hematocrit (HCT), circulating RBC volume is more difficult to measure. Thus, the HCT is often used in RBC transfusion decisions. However, several previous studies of low birth weight infants have reported that the correlation between HCT and circulating RBC volume is poor. Using a robust nonradioactive method based on in vivo dilution of biotinylated RBC enumerated by flow cytometry, the present study reexamined the correlation between HCT and circulating RBC volume in very low birth weight infants. Venous and capillary HCT levels were compared with circulating RBC volume measured using the biotin method. Twenty-six stable very low birth weight infants with birth weights less than 1300 g were studied on 43 occasions between 7 and 79 d of life. Venous HCT values correlated highly with circulating RBC volume (r = 0.907;p < 0.0001). However, the mean 95% confidence limits for prediction of circulating RBC volume from venous HCT (the average error of prediction) was ±13.4 mL/kg. The correlation between HCT and circulating RBC volume is strong in older stable very low birth weight infants. However, clinically important uncertainty exists in estimating circulating RBC volume and the associated RBC transfusion needs of an individual infant based on venous HCT. Because direct measurement of circulating RBC volume is not yet practical, the HCT (or the blood Hb concentration) remains the best available indirect indicator.

RBCs labeled at two biotin densities permit simultaneous and repeated measurements of circulating RBC volume

BACKGROUND: The extend potential applications of a nonradioactive method for measuring circulating RBC volume, we tested the hypothesis that RBC volume could be determined independently using two populations of RBCs labeled with low‐density biotin (LDB1) and high‐density biotin (HDB).

Circulating red cell volume and red cell survival can be accurately determined in sheep using the [14C]cyanate label.

The sheep commonly serves as an animal model for investigation of human fetal and newborn erythropoiesis and red blood cell kinetics. Measurement of red cell volume (RCV) and survival (RCS) in sheep would be useful for studying mechanisms of neonatal anemia. Unfortunately 51Cr, the standard method for RCV, is not suitable for RCS in sheep because 51Cr leaves the red cell too rapidly. We developed and validated the permanent label[14C]cyanate as a method for measuring both RCV and RCS in sheep. In 19 sheep, RCV was determined simultaneously using [14C]cyanate and51 Cr. RCV determined by [14C]cyanate agreed almost perfectly with RCV by 51Cr; correlation coefficient = 0.990. The line of regression had a slope of 0.94 and an intercept of 40; these parameters are not significantly different from a line of identity. In nine sheep, RCS was determined using [14C]cyanate. Survival after d 1 accurately fit a model containing two components: 1) an early exponential loss likely related to damage caused by labeling and handling and 2) a linear decrease that reflected normal survival of undamaged red cells. Mean potential life span (MPL) determined from the linear phase was 114 ± 12 d (mean± 1 SD). These results agree with reported MPL values determined either by 59Fe or differential hemolysis. Together, these observations establish [14C]cyanate-labeled red cells as a tool for measuring both RCV and RCS in sheep and enhance the value of the ovine model for investigating neonatal anemia.

A study of the interaction of avidin with 2-anilinonaphthalene-6-sulfonic acid as a probe of the biotin binding site

The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosowers Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.

Red cell volume can be accurately determined in sheep using a nonradioactive biotin label.

The sheep has served as an informative animal model for investigation of human fetal and newborn erythropoiesis and red blood cell (RBC) kinetics. We previously validated the permanent label (14C)cyanate for measuring red cell volume (RCV) in sheep. Here, we validate biotin labeling of RBCs as a nonradioactive method for measuring RCV in sheep with the anticipation that it can be applied in studies of human infants. The RCV was determined simultaneously using two techniques for quantitation of the biotin label. The first one quantified total blood concentration of biotin label on biotin-labeled RBCs using (125I)streptavidin. The second one enumerated biotin-labeled RBCs by flow cytometry after incubation with fluorescein-conjugated avidin. RCV measurements made using the two biotin quantitation techniques were validated against both (14C)cyanate and 51Cr as reference methods. Both biotin techniques produced RCV values that agreed well with the reference methods and with each other, producing correlation coefficients averaging ≥0.93. Sequential repetitive measurements in the same animal also agreed with the (14C)cyanate method and each other (average difference <10%). These results establish biotin-labeled RBCs as an accurate method for performing RCV measurements in sheep. This biotin method can be applied in studies that model neonatal erythropoiesis.

[14C]Cyanate Labeling of Sheep Red Cells: Covalent Binding to Hemoglobin Continues in Vivo for a Day

The sheep is a useful model to study fetal and newborn physiology including perinatal erythropoiesis and red cell kinetics. A practical, economical method for measuring red cell survival (RCS) in sheep would be very valuable. However, 51Cr is unsatisfactory, and suitable alternatives have not been published. In the course of investigating [14C]cyanate as a label for sheep red cells, we observed continued covalent labeling over 24 h in vivo that was great enough to introduce a substantial artifact into two commonly used parameters of RCS: posttransfusion recovery(PTR24) and time to 50% decrease (T50) when referenced to time zero. In a simulation of in vivo conditions, the amount of14 C bound to Hb increased 26 ± 6% (mean ± 1 SD,n = 11) over 24 h. To investigate the mechanism of the increasing14 C bound, acid-acetone extraction, molecular sieve chromatography, and density gradient separation were used separately or in combination to quantitate intracellular free 14C and 14C covalently bound to intracellular proteins. Free 14C decreased as protein-bound[14C]cyanate increased. These studies provide evidence that covalent binding of [14C]cyanate to intracellular Hb continues in vivo for the first 24 h and that the source of the increase is intracellular free[14C]cyanate. We conclude that 1) PTR24 cannot be accurately determined by [14C]cyanate unless labeled red cells are incubated before infusion to allow the cyanate reaction to approach completion and 2) RCS by [14C]cyanate should be referenced to blood concentrations at 24 h.

Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked by a biotin label.

BACKGROUND: Safe, accurate methods to reliably measure circulating red blood cell (RBC) kinetics are critical tools to investigate pathophysiology and therapy of anemia, including hemolytic anemias. This study documents the ability of a method using biotin‐labeled RBCs (BioRBCs) to measure RBC survival (RCS) shortened by coating with a highly purified monomeric immunoglobulin G antibody to D antigen.