Use of bi-level biotinylation for concurrent measurement of in vivo recovery and survival in two rabbit platelet populations.

BACKGROUND: The objectives of this research were 1) to determine whether two populations of platelets may be labeled with different levels of biotin and followed concurrently in vivo by flow cytometry and 2) to determine whether the level of biotinylation affects the in vivo platelet recovery and survival. STUDY DESIGN AND METHODS: Two platelet aliquots were biotinylated under conditions that resulted in either a lower or a higher number of biotin molecules per platelet. After transfusion, the two populations were distinguished and quantitated by flow cytometry. RESULTS: In five animals, recoveries were 69.8 +/- 27.0 percent for low-biotin platelets and 72.6 +/- 26.7 percent for high-biotin platelets. For each animal, the recoveries agreed closely. Life span, determined by the multiple-hit method, was 2.68 +/- 0.63 days for low-biotin platelets and 2.58 +/- 0.69 days for high-biotin platelets. These values for recovery and life span are consistent with those measured in rabbits by using radioisotope labels. CONCLUSION: Platelet biotinylation offers a nonisotopic method for direct comparison of alternative harvest and storage conditions. It also offers the potential for simultaneous evaluation of the in vivo characteristics of platelets from at least two donors.

Fetal hemoglobin and potassium in isolated transferrin receptor-positive dense sickle reticulocytes.

A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.

Inhibition of sickling after reduction of intracellular hemoglobin concentration with an osmotic pulse: characterization of the density and hemoglobin concentration distributions.

Hemoglobin S polymerization is markedly dependent on intracellular hemoglobin concentration. In the studies presented here, sickle RBC were subjected to a transient osmotic stress, which induced a short period of increased membrane permeability and allowed partial efflux of Hb S. Morphological sickling of the resulting hypochromic RBC was inhibited. The response of RBC to this osmotic pulse is influenced by the presence of a polyanion, which in these experiments was either inositol hexaphosphate (IHP, 27 mM or 46 mM) or pyrophosphate (69 mM or 95 mM). The decrease in MCHC, measured manually, ranged from 3.1 +/- 1.7 (1 SD) to 6.3 +/- 2.8 g/dl, depending on the conditions used during modification. Parallel electronic analysis of RBC indices demonstrated a comparable decrease in MCHC which was due to both an increased MCV and a decreased MCH. Since the modified cell population is quite heterogeneous, cells were analyzed using discontinuous stractan gradients and/or a laser-based instrument which measures the hemoglobin concentration (HC) of individual cells. For most treatment conditions, the modified cells have a bimodal HC distribution with one peak centered at about 20 g/dL and the other peak corresponding to the unmodified cells. With the higher concentration of IHP, however, many cells had an intermediate HC. For modified RBC with a bimodal HC distribution (27 mM IHP, 69 mM PP, 95 mM PP), inhibition of morphological sickling was proportional to the change in HC and there were no subpopulations with an increased tendency to undergo sickling. However, the intermediate density cells present when RBCs were treated with the higher concentration of IHP underwent sickling at a higher oxygen partial pressure than control cells.

Studies with biotinylated RBC: (1) use of flow cytometry to determine posttransfusion survival and (2) isolation using streptavidin conjugated magnetic beads.

Methods are reported for the quantitation and isolation of biotinylated red blood cells (B- RBC). The first method is for determination of posttransfusional survival of rabbit RBC by flow cytometry. The survival of B-RBC was measured using both fresh and paraformaldehyde-fixed cells with similar results. The posttransfusion survival of rabbit RBC measured in this way was normal. There was no indication of increased cell destruction due to antibodies directed against B-RBC and no evidence for loss of biotin from circulating cells. The second methodology is for the isolation of B-RBC from blood with streptavidin-coated magnetic beads. At least eighty percent of positive cells were recovered with very few false positives. Both methods may be helpful in the study of resealed erythrocytes.

Sickle cells modified by an osmotic pulse in the presence of inositol hexaphosphate have decreased intracellular hemoglobin concentration and decreased in vitro sickling without prolonged in vivo survival.

An osmotic pulse was used to modify red blood cells (RBC) from two patients with sickle cell disease, resulting in an increased volume and decreased hemoglobin content. This treatment yielded cells which were divided into two populations, one in which RBC had markedly decreased hemoglobin concentration and another in which the cells appeared to be unmodified. Morphological sickling at low oxygen partial pressure was markedly decreased. However, there was no evidence for increased RBC lifespan when the 51Cr-labeled, modified cells were reinfused.