Superior survival of ex vivo cultured human reticulocytes following transfusion into mice.

The generation of cultured red blood cells from stem cell sources may fill an unmet clinical need for transfusion-dependent patients, particularly in countries that lack a sufficient and safe blood supply. Cultured red blood cells were generated from human CD34+ cells from adult peripheral blood or cord blood by ex vivo expansion, and a comprehensive in vivo survival comparison with standard red cell concentrates was undertaken. Significant amplification (>105-fold) was achieved using CD34+ cells from both cord blood and peripheral blood, generating high yields of enucleated cultured red blood cells. Following transfusion, higher levels of cultured red cells could be detected in the murine circulation compared to standard adult red cells. The proportions of cultured blood cells from cord or peripheral blood sources remained high 24 hours post-transfusion (82±5% and 78±9%, respectively), while standard adult blood cells declined rapidly to only 49±9% by this time. In addition, the survival time of cultured blood cells in mice was longer than that of standard adult red cells. A paired comparison of cultured blood cells and standard adult red blood cells from the same donor confirmed the enhanced in vivo survival capacity of the cultured cells. The study herein represents the first demonstration that ex vivo generated cultured red blood cells survive longer than donor red cells using an in vivo model that more closely mimics clinical transfusion. Cultured red blood cells may offer advantages for transfusion-dependent patients by reducing the number of transfusions required.

Erythrocyte antigen and reticulocyte engraftment after allogeneic hematopoietic stem cell transplantation.

The aim of this study was to study the usefulness of erythrocyte antigen (EA) measurement to study engraftment after allogeneic HSCT. In all, 31 consecutive patients receiving HLA-identical bone marrow (BM) (n=13) or peripheral blood stem cells (n=18) were investigated. Apart from the ABO group, 15 EAs representing six minor blood groups were followed by the simple tube agglutination technique. A total of 20 (64.5%) patients received ABO-identical, eight (25.8%) received ABO minor and three (9.7%) received ABO major mismatched grafts. In all, 29 patients were followed for a median of 12 (6-16) months; 65% of the patients expressed donor type EA 1 month and almost all did so 6 months after transplant. Reticulocyte engraftment was significantly shorter than EA engraftment (median 18 vs 35 days) (P=0.001). Patients who received PB stem cells showed significantly faster EA and reticulocyte engraftment than patients who received BM stem cells (P=0.038 and 0.025). ABO compatibility did not have an impact on reticulocyte and EA engraftment (P=0.4 and 0.55). The earliest donor type EA detected was from the Rh and Kidd system. These data suggest that EA and reticulocyte assays are useful in monitoring engraftment.

In vitro and in vivo persistence of reticulocytes from donor red cells.

BACKGROUND: Reticulocytes are important in the phenotyping of transfused patients. Reticulocytes can persist in blood units for the shelf life of the unit. STUDY DESIGN AND METHODS: Temperature dependence of reticulocyte persistence was examined in vitro at 4, 24, and 37 degrees C by using thiazole orange staining and flow cytometric analysis. Two-color flow cytometric analysis was used to evaluate the persistence of donor reticulocytes in transfused patients. RESULTS: Flow cytometric analysis using thiazole orange demonstrated that persistence of reticulocytes in units of stored CPDA-1 blood was temperature-dependent. Reticulocytes disappeared over 13 and 6 days at 24 degrees C and 37 degrees C, respectively, but at 4 degrees C the reticulocyte count changed little over 35 days. Two-color flow cytometric analysis of reticulocyte antigens was used to follow donor reticulocytes in 14 transfusion events in nine different patients. Donor reticulocytes persisted through 24 hours in 75 percent of the patients and were detectable at 48 hours in three patients. CONCLUSION: This study demonstrates that reticulocytes persist during refrigerated storage; they are detectable in the circulation of most recipients for the first 24 hours after transfusion and in the circulation of a few recipients after 48 hours. These findings may have relevance for separation techniques based on reticulocyte density in samples drawn shortly after transfusion and for evaluation of reticulocyte counts in patients with hematologic abnormalities.

Does the Pb(2+)-catalyzed depolymerization of RNA occur in vivo.

In order to determine if Pb2+ depolymerizes RNA, reticulocytes were transfused into rabbits that had been dosed with lead acetate and also into untreated controls. The messenger RNA from these reticulocytes was the examined to determine if in vivo exposure to the Pb2+ had impaired the integrity of the mRNA. The total amount of mRNA did not vary between controls and Pb(2+)-treated rabbits. However, when the ability of the mRNA to program protein synthesis in cell-free hemolysates was tested, a marked loss in biological activity of the mRNA from the Pb(2+)-treated rabbits was observed. That the loss in biological activity was due to the depolymerization of the mRNA in vivo was shown by an increase in the number of free 5' hydroxyl groups in the poly (A+) RNA from the Pb(2+)-treated rabbits. The ratio of polyribosomes to monoribosomes was also decreased. In vitro experiments on the effects of Pb2+ on globin mRNA were also performed and showed that at micromolar Pb2+ concentrations the mRNA was attacked in the vicinity of the 5' end with little damage to the rest of the molecule. These data show that Pb2+ catalyzes cleavage of phosphodiester bonds resulting in loss of template activity of mRNA in vivo as well as in vitro.

Effect of exchange transfusion of reticulocytes on in vitro and in vivo intestinal iron (Fe3+) absorption in mice.

The effect of acute changes in erythron demands and in plasma-iron turnover (PIT) on the in vitro and in vivo absorption of 59Fe3+ was studied in the mouse. Hypoxic exposure for 20 h (a time at which intestinal iron absorption is markedly stimulated) induced reticulocytosis with a marked elevation in PIT. More prolonged exposure (3 d) further enhanced the plasma-iron clearance, even though the absorption of 59Fe was not further increased. Recipient mice, exchange transfused with whole blood from phenylhydrazine-treated animals, had a marked reticulocytosis and elevated PIT. However, in vivo studies exhibited only a small enhancement in intestinal 59Fe absorption. In vitro studies, in contrast, showed no changes in the kinetic parameters for duodenal Fe3+ uptake in similarly-transfused mice. Blood cells, rather than plasma, were responsible for the enhanced in vivo absorption in the transfused animals. These data indicate that acute changes in body iron demands and in PIT have only a small effect on iron absorption via a process independent of the adaptive increase in carrier-mediated uptake following chronic (3 d) hypoxia. This regulatory process, however, is inadequate to explain the adaptive changes seen during acute (20 h) hypoxic exposure.

Effect of transfused reticulocytes on iron exchange.

A animal model was developed whereby reticulocyte-rich blood was introduced into normal rats by exchange transfusion. Measurements of plasma iron turnover was made at similar plasma iron concentrations before and after exchange transfusions. High reticulocyte blood obtained from animals rendered iron deficient by diet or by treatment with phenylhydrazine resulted in a mean increase of 86% in internal iron exchange, while the plasma iron turnover was unaffected by exchange with normal red cells. Since iron input from reticuloendothelial cells could have increased due to breakdown of transfused cells, iron absorption was also measured. Within 1 hr and for a least 6 hr after exchange with high reticulocyte blood, mean absorption in six groups of animals was increased over control animals by 50%-130%. The increased plasma iron turnover and absorption was not mediated by a decrease in plasma iron or an increase in unsaturated iron-binding capacity. Indeed, a higher plasma iron and transferrin saturation augmented the movement of iron into the plasma from iron-donating tissues. It is proposed that the donation of iron by transferrin in some way immediately facilitates the procurement of more iron by transferrin.

Laboratory studies of erythrocytic pyruvate kinase deficiency. Pathogenesis of the hemolysis.

Genetic polymorphism exists in erythrocytic pyruvate kinase (PK)-deficient hemolytic anemia, as briefly described. Destruction of erythrocytes varied in extent, but its mechanisms in different PK-deficient polymorphic persons investigated were similar. A paradoxical post-splenectomy reticulocytosis regularly occurred. Qualitative enzymatic differences, the biochemistry, and measurements of erythrocytic destruction were made in several PK variants. 51Cr autologous and cross-transfusions of PK-deficient erythrocytes into volunteers showed multimodel regression lines of several erythrocytic cohorts. 59Felabeled PK-deficient reticulocytes donated by PK-deficient splenectomized subjects were transfused 20 hours before splenectomy into two PK-deficient infants with hemolysis, and into two adult volunteers with autoimmune thrombocytopenia. The highest reticulocyte concentration in any organ initially was within the spleen. Radioiron-labeled erythrocytes and cytologic data showed large splenic reticulocyte pools. Splenic macrophages ingesting reticulocytes and erythrocytes were seen by both light and electron microscopy of the splenic pulp. After cyanide additives inhibiting reticulocyte oxidative phosphorylation, a bizarre erythrocytic cytologic configuration was found by scanning electron microscopy. These studies of PK-mutant subjects with PK-deficient erythrocytic hemolysis showed age dependent destruction of erythrocytes. Bimodal Cr survival data suggested reticuloendothelial removal of short-lived erythrocytes and reticulocytes. The transfusions of radioironlabeled PK reticulocytes and the data obtained by scanning electron microscopy suggested that the spleen was the initial hostile organ destroying a cohort of susceptible erythrocytes, prinicpally reticulocytes.

Effects of short-term epithelial reticular cell and whole organ thymus grafts in neonatally thymectomized mice.

Neonatally thymectomized mice were implanted with thymus grafts composed of epithelial reticular cells for periods of 7 and 14 days. Regardless of whether the grafts were placed immediately after thymectomy, or at 3 wk of age, there was little recovery of the lymphocyte depletion and impaired immunologic responsiveness, characteristically found in a neonatally thymectomized host. The findings were similar in animals studied at 2 months or 2 wk after graft removal. Many of the short-term remnant grafts were populated with lymphocytes and had attained the morphologic appearance of thymus by 14 days. A lesser degree of lymphocyte depletion and impaired responsiveness to SRBC occurred if thymectomy was delayed until 7 days of age, if remnant grafts were removed after 2 months, and if intact neonatal thymus was used for the short-term grafts. Complete normality was found in some of the animals in all of these groups. These observations suggest a direct role for mature thymus lymphocytes in reconstituting the neonatally thymectomized host and indicate that epithelial cell function is to direct the maturation of cells that ultimately behave as thymus lymphocytes.