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Background: In hematologic and transfusion medicine research, measurement of red blood cell (RBC) in vivo kinetics must be safe and accurate. Recent reports indicate use of biotin-labeled RBC (BioRBC) to determine red cell survival (RCS) offers substantial advantages over 51 Cr and other labeling methods. Occasional induction of BioRBC antibodies has been reported., Study Design and Methods: To investigate the causes and consequences of BioRBC immunization, we reexposed three previously immunized adults to BioRBC and evaluated the safety, antibody emergence, and RCS of BioRBC., Results: BioRBC re-exposure caused an anamnestic increase of plasma BioRBC antibodies at 5-7 days; all were subclass IgG 1 and neutralized by biotinylated albumin, thus indicating structural specificity for the biotin epitope. Concurrently, specific antibody binding to BioRBC was observed in each subject. As biotin label density increased, the proportion of BioRBC that bound increased antibody also increased; the latter was associated with proportional accelerated removal of BioRBC labeled at density 6 μg/mL. In contrast, only one of three subjects exhibited accelerated removal of BioRBC density 2 μg/mL. No adverse clinical or laboratory events were observed. Among three control subjects who did not develop BioRBC antibodies following initial BioRBC exposure, re-exposure induced neither antibody emergence nor accelerated BioRBC removal., Discussion: We conclude re-exposure of immunized subjects to BioRBC can induce anamnestic antibody response that can cause an underestimation of RCS. To minimize chances of antibody induction and underestimation of RCS, we recommend an initial BioRBC exposure volume of ≤10 mL and label densities of ≤18 μg/mL., (© 2022 AABB.)

The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-51 ( 51 Cr) RBC labeling for determining RBC volume, 24-hour posttransfusion RBC recovery, and long-term RBC survival. Here we provide evidence supporting adoption of a method for kinetics that uses the biotin-labeled RBCs (BioRBCs) as a superior, versatile method for both regulatory and investigational purposes. RBC kinetic analysis using BioRBCs has important methodologic, analytical, and safety advantages over 51 Cr-labeled RBCs. We critically review recent advances in labeling human RBCs at multiple and progressively lower biotin label densities for concurrent, accurate, and sensitive determination of both autologous and allogeneic RBC population kinetics. BioRBC methods valid for RBC kinetic studies, including successful variations used by the authors, are presented along with pharmacokinetic modeling approaches for the accurate determination of RBC pharmacokinetic variables in health and disease. The advantages and limitations of the BioRBC method-including its capability of determining multiple BioRBC densities simultaneously in the same individual throughout the entire RBC life span-are presented and compared with the 51 Cr method. Finally, potential applications and limitations of kinetic BioRBC determinations are discussed., (© 2018 AABB.)

Background: Critically ill preterm very-low-birthweight (VLBW) neonates (birthweight ≤ 1.5 kg) frequently develop anemia that is treated with red blood cell (RBC) transfusions. Although RBCs transfused to adults demonstrate progressive decreases in posttransfusion 24-hour RBC recovery (PTR 24 ) during storage-to a mean of approximately 85% of the Food and Drug Administration-allowed 42-day storage-limited data in infants indicate no decrease in PTR 24 with storage., Study Design and Methods: We hypothesized that PTR 24 of allogeneic RBCs transfused to anemic VLBW newborns: 1) will be greater than PTR 24 of autologous RBCs transfused into healthy adults and 2) will not decrease with increasing storage duration. RBCs were stored at 4°C for not more than 42 days in AS-3 or AS-5. PTR 24 was determined in 46 VLBW neonates using biotin-labeled RBCs and in 76 healthy adults using 51 Cr-labeled RBCs. Linear mixed-model analysis was used to estimate slopes and intercepts of PTR 24 versus duration of RBC storage., Results: For VLBW newborns, the estimated slope of PTR 24 versus storage did not decrease with the duration of storage (p = 0.18) while for adults it did (p < 0.0001). These estimated slopes differed significantly in adults compared to newborns (p = 0.04). At the allowed 42-day storage limit, projected mean neonatal PTR 24 was 95.9%; for adults, it was 83.8% (p = 0.0002)., Conclusions: These data provide evidence that storage duration of allogeneic RBCs intended for neonates can be increased without affecting PTR 24 . This conclusion supports the practice of transfusing RBCs stored up to 42 days for small-volume neonatal transfusions to limit donor exposure., (© 2017 AABB.)

Background: Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dosing occasionally induces antibodies, a sensitive and specific anti-BioRBC detection assay is needed., Study Design and Methods: Aims were to 1) develop a gel card assay to evaluate existing, naturally occurring and BioRBC-induced plasma antibodies, 2) compare gel card and tube agglutination detection results, and 3) test for a relationship of antibody induction and BioRBC dose. Reagent BioRBCs were prepared using sulfo-NHS biotin ranging from densities 18 (BioRBC-18) to 1458 (BioRBC-1458) µg/mL RBCs., Results: Among BioRBC-exposed subjects, gel card and tube agglutination results were concordant in 21 of 22 adults and all 19 infant plasma samples. Gel card antibody detection sensitivity was more than 10-fold greater than tube agglutination. Twelve to 16 weeks after BioRBC exposure, induced anti-antibodies were detected by gel card in three of 26 adults (12%) at reagent densities BioRBC-256 or less, but in none of 41 infants. Importantly, induced anti-BioRBC antibodies were associated with higher BioRBC dose (p = 0.008); no antibodies were detected in 18 subjects who received BioRBC doses less than or equal to BioRBC-18. For noninduced BioRBC antibodies, six of 1125 naïve adults (0.3%) and none of 46 naïve infants demonstrated existing anti-BioRBC antibodies using reagent BioRBC-140 or -162. Existing anti-BioRBCs were all neutralized by biotin compounds, while induced antibodies were not., Conclusions: The gel card assay is more sensitive than the tube agglutination assay. We recommend reagent BioRBC-256 for identifying anti-BioRBCs. Use of a low total RBC biotin label dose (≤ BioRBC-18) may minimize antibody induction., (© 2017 AABB.)

Direct measurement of red blood cell (RBC) survival in humans has improved from the original accurate but limited differential agglutination technique to the current reliable, safe, and accurate biotin method. Despite this, all of these methods are time consuming and require blood sampling over several months to determine the RBC lifespan. For situations in which RBC survival information must be obtained quickly, these methods are not suitable. With the exception of adults and infants, RBC survival has not been extensively investigated in other age groups. To address this need, we developed a novel, physiology-based mathematical model that quickly estimates RBC lifespan in healthy individuals at any age. The model is based on the assumption that the total number of RBC recirculations during the lifespan of each RBC (denoted by N max) is relatively constant for all age groups. The model was initially validated using the data from our prior infant and adult biotin-labeled red blood cell studies and then extended to the other age groups. The model generated the following estimated RBC lifespans in 2-year-old, 5-year-old, 8-year-old, and 10-year-old children: 62, 74, 82, and 86 days, respectively. We speculate that this model has useful clinical applications. For example, HbA1c testing is not reliable in identifying children with diabetes because HbA1c is directly affected by RBC lifespan. Because our model can estimate RBC lifespan in children at any age, corrections to HbA1c values based on the model-generated RBC lifespan could improve diabetes diagnosis as well as therapy in children.

Background: Critically ill preterm and term neonates are at high risk for negative iron balance due to phlebotomy that occurs with frequent laboratory monitoring, and the high iron demand of rapid growth. Understanding the prioritization of iron between red blood cells (RBCs) and brain is important given iron's role in neurodevelopment., Methods: Ten neonatal twin lamb pairs (n = 20) underwent regular phlebotomy for 11 d. Lambs were randomized to receive no iron or i.v. daily iron supplementation from 1 to 5 mg/kg. Serum hemoglobin concentration and reticulocyte count were assayed, iron balance calculated, and iron content of RBCs, liver, brain, muscle, and heart measured at autopsy., Results: Among phlebotomized lambs: (i) liver iron concentration was directly related to net iron balance (r = 0.87; P < 0.001) and (ii) brain iron concentration was reduced as a function of net iron balance (r = 0.63) only after liver iron was depleted. In animals with negative iron balance, total RBC iron was maintained while brain iron concentration decreased as a percentage of the iron present in RBCs (r = -0.70; P < 0.01) and as a function of reticulocyte count (r = -0.63; P < 0.05)., Conclusion: Phlebotomy-induced negative iron balance limits iron availability to the developing brain.

The lifespan of red blood cells (RBCs) plays an important role in the study and interpretation of various clinical conditions. Yet, confusion about the meanings of fundamental terms related to cell survival and their quantification still exists in the literature. To address these issues, we started from a compartmental model of RBC populations based on an arbitrary full lifespan distribution, carefully defined the residual lifespan, current age, and excess lifespan of the RBC population, and then derived the distributions of these parameters. For a set of residual survival data from biotin-labeled RBCs, we fit models based on Weibull, gamma, and lognormal distributions, using nonlinear mixed effects modeling and parametric bootstrapping. From the estimated Weibull, gamma, and lognormal parameters we computed the respective population mean full lifespans (95 % confidence interval): 115.60 (109.17-121.66), 116.71 (110.81-122.51), and 116.79 (111.23-122.75) days together with the standard deviations of the full lifespans: 24.77 (20.82-28.81), 24.30 (20.53-28.33), and 24.19 (20.43-27.73). We then estimated the 95th percentiles of the lifespan distributions (a surrogate for the maximum lifespan): 153.95 (150.02-158.36), 159.51 (155.09-164.00), and 160.40 (156.00-165.58) days, the mean current ages (or the mean residual lifespans): 60.45 (58.18-62.85), 60.82 (58.77-63.33), and 57.26 (54.33-60.61) days, and the residual half-lives: 57.97 (54.96-60.90), 58.36 (55.45-61.26), and 58.40 (55.62-61.37) days, for the Weibull, gamma, and lognormal models respectively. Corresponding estimates were obtained for the individual subjects. The three models provide equally excellent goodness-of-fit, reliable estimation, and physiologically plausible values of the directly interpretable RBC survival parameters.

Fetal RBCs are produced during a period of very rapid growth and stimulated erythropoiesis under hypoxic intrauterine conditions. Fetal RBC life span varies with gestational age (GA) and is shorter than that in healthy adults. Due to the special kinetic properties of life span-based survival of human RBCs, a mathematical model-based kinetic analysis of the survival of fetal RBCs shortly after birth provides a unique opportunity to "look backward in time" to evaluate fetal erythropoiesis. This work introduces a novel method that utilizes postnatal in vivo RBC survival data collected within 2 days after birth to study both nonsteady-state (non-SS) in utero RBC production and changing fetal RBC life span over time. The effect of changes in erythropoiesis rate and RBC life span and the effect of multiple postnatal phlebotomies on the RBC survival curves were investigated using model-based simulations. This mathematical model, which considers both changes in the rate of erythropoiesis and RBC life span and which accurately accounts for the confounding effect of multiple phlebotomies, was applied to survival curves for biotin-labeled RBCs from ten anemic very low birth weight preterm infants. The estimated mean fetal RBC production rate scaled by body weight was 1.07 × 10(7) RBCs/day g, and the mean RBC life span at birth was 52.1 days; these values are consistent with reported values. The in utero RBC life span increased at a rate of 0.51 days per day of gestation. We conclude that the proposed mathematical model and its implementation provide a flexible framework to study in utero non-SS fetal erythropoiesis in newborn infants.

Background: The quality of transfused red blood cells (RBCs) to treat anemia depends on its potential for oxygen delivery, governed by two properties: 1) initial posttransfusion recovery and 2) life span of initially surviving RBCs. The latter property is poorly evaluated by the traditional mean potential life span (MPL) or mean cell age (MA), because these parameters do not evaluate how long transfused RBCs remain in circulation. Furthermore, evaluation of MPL is based on two problematic assumptions regarding transfused RBCs: 1) they were produced at a constant steady-state rate and 2) they have similar storage life spans., Study Design and Methods: This work introduces a new parameter, the mean remaining life span (MRL) to quantify transfused RBC survival (TRCS) and presents a simple algorithm for its evaluation. The MRL was calculated for four adult subjects with sickle cell disease and four adult diabetic and nondiabetic subjects using RBC survival data sets with existing TRCS parameters., Results: The RBC survival curves in the sickle cell subjects were nonlinear with rapid decline in survival within the first 5 days. The MRL was approximately 4.6 days. Thus, the MRL was indicative of the survival of all transfused RBCs. For the diabetic and nondiabetic subjects, the RBC disappearance curves did not deviate substantially from a linear decline. Thus, the estimates for MRL ranging from 39 to 51 days are similar to the MA previously computed., Conclusion: MRL overcomes limitations of previously proposed TRCS parameters, is simpler to calculate, and is physiologically and clinically more appropriate., (© 2014 AABB.)

The goal of this review is to summarize and critically assess information concerning the biotin method to label red blood cells (RBC) for use in studies of RBC and transfusion biology-information that will prove useful to a broad audience of clinicians and scientists. A review of RBC biology, with emphasis on RBC senescence and in vivo survival, is included, followed by an analysis of the advantages and disadvantages of biotin-labeled RBC (BioRBC) for measuring circulating RBC volume, posttransfusion RBC recovery, RBC life span, and RBC age-dependent properties. The advantages of BioRBC over (51)Cr RBC labeling, the current reference method, are discussed. Because the biotin method is straightforward and robust, including the ability to follow the entire life spans of multiple RBC populations concurrently in the same subject, BioRBC offers distinct advantages for studying RBC biology and physiology, particularly RBC survival. The method for biotin labeling, validation of the method, and application of BioRBCs to studies of sickle cell disease, diabetes, and anemia of prematurity are reviewed. Studies documenting the safe use of BioRBC are reviewed; unanswered questions requiring future studies, remaining concerns, and regulatory barriers to broader application of BioRBC including adoption as a new reference method are also presented., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Anemia, a common condition among critically ill premature infants, is affected by red blood cell (RBC) survival (RCS). We hypothesized that transfused allogeneic Kidd antigen-mismatched RBCs would demonstrate the same concurrent RCS tracking as RBCs multilabeled at separate, discrete low densities with biotin (BioRBCs)., Methods: Allogeneic RBCs from adult donors were labeled at four biotin densities, mixed, and transfused into 17 anemic premature infants. Nine of the donors and neonates were Kidd antigen mismatched. Serial posttransfusion blood samples were assayed for up to 8 wk by flow cytometry to track the survival of the proportions of Kidd antigen-mismatched and Kidd antigen-biotinylated RBCs., Results: Using linear mixed modeling to compare results, RCS of the three lowest BioRBC densities was similar to RCS by Kidd antigen mismatch and to one another. RCS of RBCs labeled at the highest BioRBC density was shortened., Conclusion: RCS of different populations of RBCs can be tracked concurrently and reliably using the three lowest BioRBC densities. Although comparable RCS results can be achieved using Kidd antigen mismatches, BioRBCs are preferred for investigating neonatal anemia because biotin labeling of both allogeneic and autologous RBCs is possible.

Background: Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology and treatment of anemia. Our objective was to validate the multidensity biotin method for RCS determination in sheep, a commonly used model of RBC physiology. [(14) C]Cyanate served as the reference method for long-term RCS because the (51) Cr method (the reference method for humans) is not reliable in sheep., Study Design and Methods: Aliquots of autologous RBCs from eight adult sheep were labeled with [(14) C]cyanate and four separate densities of biotin (BioRBCs) and reinfused. Short-term RCS was assessed by posttransfusion recovery at 24 hours (PTR(24) ); long-term RCS was assessed by the time to 50% survival (T(50) ) and mean potential life span (MPL)., Results: Values for PTR(24) of the four BioRBC densities were not different. Values for RCS as reflected by T(50) and MPL were nearly identical for [(14) C]cyanate and the two intermediate-density BioRBC populations. In contrast, the lowest-density BioRBC population survived slightly longer (p < 0.01), but with a difference of no clinical significance. The highest-density BioRBC population importantly shortened RCS (p < 0.01 compared to the two intermediate densities)., Conclusion: This study provides evidence that BioRBCs labeled at four biotin densities can be used to independently and simultaneously measure short-term RCS and that BioRBCs labeled at the three lowest biotin densities can be used to accurately and simultaneously measure long-term RCS. Because the sheep RBC model is comparable to humans, this nonradioactive method has promise for use in RBC kinetic studies in neonates and pregnant women., (© 2012 American Association of Blood Banks.)

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., Study Design and Methods: Autologous RBCs from 10 healthy D+ subjects were labeled with either biotin or (51) Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for (51) Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR(24) ) for short-term RCS; 2) time to 50% decrease of the label (T(50) ), and 3) mean potential life span (MPL) for long-term RCS., Results: BioRBCs tracked both normal and shortened RCS accurately relative to (51) Cr. For lightly coated RBCs, mean PTR(24) , T(50) , and MPL results were not different between BioRBCs and (51) Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR(24) by BioRBCs (84 ± 18%) was not different from (51) Cr (81 ± 10%); mean T(50) by BioRBCs (23 ± 17 days) was not different from (51) Cr (22 ± 18 days)., Conclusion: RCS shortened by coating with anti-D can be accurately measured by BioRBCs. We speculate that BioRBCs will be useful for studying RCS in conditions involving accelerated removal of RBCs including allo- and autoimmune hemolytic anemias., (© 2011 American Association of Blood Banks.)

Background: Safe, accurate methods permitting simultaneous and/or repeated measurement of red blood cell (RBC) survival (RCS) are important to investigate pathophysiology and therapy of anemia. Methods using chromium 51 ((51) Cr)-labeled RBCs are unacceptable for infants, children, and pregnant women. We report RCS measured in vivo using RBCs labeled with several densities of biotin (BioRBCs)., Study Design and Methods: Aliquots of autologous RBCs from eight healthy adult subjects were labeled separately at four discrete biotin densities, mixed, and infused. The proportion of each population of BioRBCs circulating was determined serially by flow cytometry over 20 weeks. For each population, RCS was assessed by the following: 1) posttransfusion BioRBC recovery at 24 hours (PTR(24) ); 2) time to decrease to 50% of the enrichment at 24 hours (T(50) ); and 3) mean potential lifespan (MPL)., Results: Among the four BioRBC densities, no significant differences in PTR(24) were observed. T(50) and MPL were similar for the two lowest BioRBC densities. In contrast, the two highest BioRBC densities demonstrated progressively decreased T(50) and MPL., Conclusions: RBCs labeled at four biotin densities can be used to independently and accurately measure PTR(24 ) and two lowest biotin densities can accurately quantitate long-term RCS. This method provides a tool for investigating anemia in infants, fetuses, and pregnant women with the following advantages over the standard (51) Cr method: 1) study subjects are not exposed to radiation; 2) small blood volumes (e.g., 20 µL) are required; and 3) multiple independent RCS measurements can be made simultaneously in the same individual., (© 2010 American Association of Blood Banks.)

Background: Anemia is a serious problem in critically ill neonates. To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin therapy, repeated measurement of red blood cell volume (RCV) and blood volume is useful. To extend our previous sheep study in which RBCs were labeled at four different biotin densities, we assessed the validity of this multidensity method for in vivo measurement of circulating RCV in humans., Study Design and Methods: In eight healthy adults, autologous RBCs were biotinylated at each of four biotin densities (6, 18, 54, and 162 µg biotinylation reagent/mL RBC), mixed, and infused intravenously; blood was sampled at 10, 20, and 60 minutes. At each time, RCV was calculated from dilution of individual RBC populations enumerated by flow cytometry. RCV measurements from the population of RBCs biotinylated at 6 µg/mL were chosen as the reference values because this density had been previously validated against the 51Cr method in vitro and in vivo in humans., Results: Values for RCVs were not significantly different among the four densities of biotinylated RBCs at any of the three time points and did not change over 60 minutes., Conclusions: These studies provide evidence that four densities of biotinylated RBCs can be used in vivo for simultaneous, independent, accurate measurements of RCV in humans. We speculate that this method will also be useful for repeated measurement of RCV and blood volume in infants and other patient populations in whom radioactive labels should be avoided., (© 2010 American Association of Blood Banks.)

Background: To investigate the pathophysiology of anemia and responses to red blood cell (RBC) transfusions and erythropoietin, repeated measurement of the circulating red blood cell volume (RCV) would be useful. Ovine erythropoiesis is similar to human erythropoiesis. Accordingly, a method for measuring RCV using either human or sheep RBCs labeled at different biotin densities has been previously validated in vitro. Here preclinical studies validating this method for in vivo measurement of circulating RCV in sheep are reported., Study Design and Methods: For each sheep, autologous RBCs were biotinylated were at four discrete densities (12, 24, 48, and 96µg biotinylation reagent/mL RBCs). The densities were mixed and infused intravenously. Blood was sampled five times over 1 hour beginning at 4 minutes. RCV values were determined based on dilution of each population of biotinylated RBCs and by the [(14) C]cyanate method., Results: There was no difference among RCVs measured at all densities through 16 minutes; however, by 60 minutes, RBCs biotinylated at the highest density overestimated RCV by 7.6%. RCV values increased 41% over the hour, consistent with equilibration with a pool of RBCs sequestered in the spleen. RCV by the [(14) C]cyanate method paralleled results by the biotin method but averaged 8% greater., Conclusions: These studies provide evidence that all four densities of biotinylated RBCs can be used in sheep to simultaneously and independently determine RCV. We speculate that the multidensity biotinylation method will be useful both for multiple simultaneous measurements and for repeated measurement of circulating RCV and blood volume in humans., (© 2010 American Association of Blood Banks.)

Because critically ill premature infants experience significant iron loss due to phlebotomy and have high iron needs for growth, Fe absorption and incorporation studies are clinically important. A prospective, controlled, randomized, open 21-d study was conducted in infants with birth weight <1300 g and gestational age < 31 wk to assess the efficacy of combining intravenous (IV) sucrose iron (Fe) with erythropoietin (EPO) for increasing Fe absorption, RBC Fe incorporation, and erythropoiesis. Three clinically stable groups were enrolled at 3-4 wk of age: Control, EPO [2100 U EPO/(kg.wk)]; and IV Fe+EPO [2 mg IV sucrose Fe/(kg.d) plus 2100 U EPO/(kg.wk)]. All subjects received 9 mg/(kg.d) of oral Fe polymaltose. Subjects were not allowed RBC transfusions. Indicators of iron status and erythropoiesis were assessed before and 18 d after treatment. On d 4, tracer doses of oral polymaltose (57)Fe and IV sucrose (58)Fe were administered, and stool and blood samples were collected for Fe absorption and incorporation determinations. Compared with the Control group, the EPO group demonstrated greater hemoglobin (Hb) concentration and reticulocyte count, but no difference in Fe incorporation. In contrast, the IV Fe+EPO group demonstrated greater total Fe incorporation, Hb concentration, plasma ferritin, and reticulocyte count compared with the Control and EPO groups. Absorption of (57)Fe and nonisotopic polymaltose Fe did not differ among the groups (range: 48-58%, and 41-47%, respectively). We conclude that IV sucrose Fe administered in combination with EPO to very-low-birth weight premature infants significantly increases RBC Fe incorporation and erythropoiesis more than EPO alone, but without increasing iron absorption.

Objective: To examine the utility of red blood cell (RBC) zinc protoporphyrin/heme ratio (ZnPP/H) as an indicator of fetal iron status, because unfavorable neurodevelopmental outcomes have been associated with poor iron status at birth, as indicated by low serum ferritin, and because few reliable indicators of fetal and early neonatal iron status exist., Methods: Consecutively studied preterm and term fetuses at delivery included the following groups: (1) control nonhypoxic, (2) fetuses with intrauterine growth retardation (IUGR), and (3) fetuses of insulin-treated mothers (FDM). We hypothesized (1) that rapid growth velocity associated with an accelerated erythropoiesis among normal fetuses will lead to reduced iron delivery to a rapidly expanding RBC mass and higher umbilical cord blood RBC ZnPP/H and (2) that fetuses that are exposed to pathologic hypoxemia will experience an additional increase in erythropoiesis and higher cord ZnPP/H. ZnPP/H was determined on saline-washed cord blood erythrocytes by hematofluorometry and was examined for its relationship with clinical factors and cord blood laboratory measurements indicative of tissue oxygenation (plasma erythropoietin [EPO] and reticulocyte count) and iron status (plasma ferritin and erythrocyte indices). Statistical testing included 1-way analysis of variance, 2-way analysis of variance with covariates, simple linear regression, and multiple regression analysis., Results: Among control group subjects, gestational age at birth was inversely correlated with RBC ZnPP/H and reticulocyte count and positively correlated with ferritin and EPO. Relative to control subjects, IUGR and FDM fetuses at specified gestational age groupings had higher ZnPP/H, lower plasma ferritin, and higher plasma EPO. Statistical modeling of the relationship between ZnPP/H and plasma ferritin among all study groups demonstrated significant impacts of gestational age, plasma EPO, maternal hypertension, and maternal smoking., Conclusions: The inverse association of fetal ZnPP/H with gestational age at birth among control subjects is attributable to erythropoietic stimulation likely as a result of increasing growth velocity at the earliest gestational ages. The relatively higher ZnPP/H observed among fetuses in the IUGR and FDM groups likely is attributable to increased erythropoietic activity secondary to pathologic hypoxemia. Decreased placental iron transfer may also have limited iron availability and contributed to elevated ZnPP/H in the IUGR group. These data support the concept that increased erythropoietic activity and/or limited iron transport may place infants of diabetic mothers and infants with growth retardation at risk for developing systemic iron deficiency later in infancy and in early childhood.

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)., Study Design and Methods: In 10 healthy adults, autologous RBCs were labeled with HDB, LDB, or 51Cr. The labeled RBCs were mixed and transfused. RBC volume was measured in postinfusion peripheral venous blood by quantitating dilution of each population of labeled RBCs., Results: RBC volume measured using either LDB or HDB cells agreed well with RBC volume measured using 51Cr. For the regression of RBC volume by LDB versus RBC volume by 51Cr, correlation = 0.994 and slope = 0.933. For HDB versus 51Cr, correlation = 0.982 and slope = 0.953. RBC volume measured a second time in four subjects with HDB agreed well; mean CV for the differences between HDB and 51Cr were less than 5 percent., Conclusions: Using RBCs labeled with two different densities of biotin, RBC volume can be accurately measured simultaneously and repeatedly in the same subject without radiation exposure.

Objective. Although many centers have introduced more restrictive transfusion policies for preterm infants in recent years, the benefits and adverse consequences of allowing lower hematocrit levels have not been systematically evaluated. The objective of this study was to determine if restrictive guidelines for red blood cell (RBC) transfusions for preterm infants can reduce the number of transfusions without adverse consequences. Design, Setting, and Patients. We enrolled 100 hospitalized preterm infants with birth weights of 500 to 1300 g into a randomized clinical trial comparing 2 levels of hematocrit threshold for RBC transfusion. Intervention. The infants were assigned randomly to either the liberal- or the restrictive-transfusion group. For each group, transfusions were given only when the hematocrit level fell below the assigned value. In each group, the transfusion threshold levels decreased with improving clinical status. Main Outcome Measures. We recorded the number of transfusions, the number of donor exposures, and various clinical and physiologic outcomes. Results. Infants in the liberal-transfusion group received more RBC transfusions (5.2 [+ or -] 4.5 [mean [+ or -] SD] vs 3.3 [+ or -] 2.9 in the restrictive-transfusion group). However, the number of donors to whom the infants were exposed was not significantly different (2.8 [+ or -] 2.5 vs 2.2 [+ or -] 2.0). There was no difference between the groups in the percentage of infants who avoided transfusions altogether (12% in the liberal-transfusion group versus 10% in the restrictive-transfusion group). Infants in the restrictive-transfusion group were more likely to have intraparenchymal brain hemorrhage or periventricular leukomalacia, and they had more frequent episodes of apnea, including both mild and severe episodes. Conclusions. Although both transfusion programs were well tolerated, our finding of more frequent major adverse neurologic events in the restrictive RBC-transfusion group suggests that the practice of restrictive transfusions may be harmful to preterm infants. Pediatrics 2005;115:1685-1691; anemia, brain, erythrocyte, preterm infants, transfusions. ABBREVIATION. RBC, red blood cell., Preterm infants are among the most heavily transfused patient populations. In 1991, it was estimated that infants with a birth weight of In recent years, efforts to reduce the number [...]

BACKGROUND: Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology and treatment of anemia. Our objective was to validate the multidensity biotin method for RCS determination in sheep, a commonly used model of RBC physiology. [14C]Cyanate served as the reference method for long-term RCS because the 51Cr method (the reference method for humans) is not reliable in sheep. STUDY DESIGN AND METHODS: Aliquots of autologous RBCs from eight adult sheep were labeled with [14C]cyanate and four separate densities of biotin (BioRBCs) and reinfused. Short-term RCS was assessed by posttransfusion recovery at 24 hours (PTR24); long-term RCS was assessed by the time to 50% survival (T50) and mean potential life span (MPL). RESULTS: Values for PTR24 of the four BioRBC densities were not different. Values for RCS as reflected by T50 and MPL were nearly identical for [14C]cyanate and the two intermediate-density BioRBC populations. In contrast, the lowest-density BioRBC population survived slightly longer (p < 0.01), but with a difference of no clinical significance. The highest-density BioRBC population importantly shortened RCS (p < 0.01 compared to the two intermediate densities). CONCLUSION: This study provides evidence that BioRBCs labeled at four biotin densities can be used to independently and simultaneously measure short-term RCS and that BioRBCs labeled at the three lowest biotin densities can be used to accurately and simultaneously measure long-term RCS. Because the sheep RBC model is comparable to humans, this nonradioactive method has promise for use in RBC kinetic studies in neonates and pregnant women. [ABSTRACT FROM AUTHOR]

BACKGROUND: The development of valid methods for repeatedly measuring red blood cell (RBC) volume (RCV) in the same individual would be useful in furthering understanding of the physiology and pathophysiology of the pregnant woman, fetus, and infant under a variety of conditions. STUDY DESIGN AND METHODS: Small volumes (5 to 100 mL) of either sheep or human blood were used to test the hypothesis that there is no significant difference in RCV and blood volume determined in vitro using as many as five populations of RBCs labeled at distinct biotin densities. By varying the mass of biotinylating reagent, the density of biotin on the surface of RBCs was incrementally increased to produce discrete populations as assessed by flow cytometric enumeration. Calculation of RCV for each biotin-labeled RBC population was based on the dilution principle. RESULTS: All biotin densities, except the most densely labeled, where variance was the greatest, accurately quantitated the in vitro blood volume to within 10 percent of the correct value. There was no bias of either overestimation or underestimation in the determination of the blood volume using either sheep or human RBCs. CONCLUSION: These in vitro results provide evidence that the multidensity biotin labeling method is sufficiently accurate to utilize in vivo for repeated determination of circulating RCV and blood volume. [ABSTRACT FROM AUTHOR]

Background. Critically ill, extremely premature infants develop anemia because of intensive laboratory blood testing and undergo multiple red blood cell (RBC) transfusions in the early weeks of life. To date, researchers have had only limited success in finding ways to reduce transfusions significantly in this patient population. Objective. To reduce RBC transfusions for these infants by using a point-of-care bedside monitor that returns analyzed blood to the patient. Design, Setting, and Patients. This was a prospective, 2-center, randomized, open, controlled, clinical trial with a 1:1 assignment of extremely low birth weight infants (weighing 500-1000 g at birth) to control or monitor groups and analysis with the intention-to-treat approach. Predefined RBC transfusion criteria were applied uniformly in the 2 groups. Interventions. Clinical treatment of study subjects with an inline, ex vivo, bedside monitor that withdraws blood through an umbilical artery catheter, analyzes blood gases and sodium, potassium, and hematocrit levels, and returns the sample to the patient. Main Outcome Measures. The total volume and number of RBC transfusions during the first 2 weeks of life and the total volume of blood removed for laboratory testing. Results. The trial was terminated prematurely when one center's NICU changed its standard method of laboratory testing. In the first 2 weeks of life, there was a nonsignificant 17% lower cumulative RBC transfusion volume in the monitor group (n = 46), compared with the control group (n = 47). However, data from the first week only (the period of greater catheter use) demonstrated a significant 33% lower cumulative RBC transfusion volume in the monitor group. Cumulative phlebotomy loss was ∼25% less in the monitor group throughout the 2-week study period. There was no difference between groups in neonatal mortality, morbidity, and neurodevelopmental outcome rates at 18 to 24 months. This is the first randomized... [ABSTRACT FROM AUTHOR]

A Hb balance model was used in very low birth weight (VLBW) infants to predict posttransfusion Hb levels from which we inferred allogeneic RBC recovery after transfusion of RBCs stored for varying periods of time. Premature VLBW infants receiving RBC transfusions during the 1st month of life were evaluated retrospectively for RBC survival of stored donor blood. Actual Hb levels measured in infant blood 1 and 2 days after RBC transfusions were com-pared to those predicted using a Hb balance model based on factors affecting blood Hb loss and gain. Transfusions were subgrouped according to whether or not infants were clinically stable at the time of RBC transfusion. Model-predicted RBC recovery was also evaluated relative to duration of RBC storage. Model-predicted mean (± SD) Hb levels 2 days after transfusion among the 30 VLBW infants receiv-ing a total of 57 RBC transfusions were only 4 percent higher than actual values observed (15.2 ± 1.2 g/dL vs. 14.7 ± 1.4, respectively; p < 0.05). The infant's clinical status at the time of transfusion did not affect predicted 1- and 2-day posttransfusion RBC recovery. Model-predicted recovery of transfused RBCs was modestly, but significantly, decreased with increasing duration of donor RBC storage (i.e., 10% lower by 42 days—the maximal allowed storage period for donor blood [p < 0.01]). Our model-predicted RBC survival results are consistent with—but not direct evidence of—hemolysis of donor blood after RBC transfusion. Although observed post-RBC Hb levels 2 days after transfusion averaged only 4 percent less than predicted, model-predicted survival of donor RBCs at 42 days suggested a modest decrease (i.e., by 10%). [ABSTRACT FROM AUTHOR]

Objective: Anemic ovine fetuses supplemented with intra-amniotic iron undergo rapid expansion of red cell mass. The present study tested the hypothesis that nonanemic fetuses that were supplemented with daily intra-amniotic iron plus intravascular injections of erythropoietin would experience accelerated erythrocyte production.Study Design: Nine late gestation ovine fetuses received 100 to 120 units of erythropoietin intravascularly plus 10 mg of iron intra-amniotically daily for 7 days (low erythropoietin dose group). Four additional fetuses received 1000 units of erythropoietin plus 10 mg iron daily for the same period (high erythropoietin dose group). Responses were compared with 9 nonsupplemented time-control fetuses. Statistical testing was by 3-factor repeated measures analysis of variance.Results: Immediately after erythropoietin injection, plasma erythropoietin concentration was elevated approximately 25- and 250-fold in the low and high erythropoietin dose groups, respectively. Erythropoietin returned to basal levels by 24 hours after the injection. Plasma iron concentration increased in the low erythropoietin dose group but not in the control or high erythropoietin dose groups. Reticulocyte index increased in both erythropoietin supplemented groups but not in control fetuses. Hematocrit level increased above control by day 5 in the low erythropoietin dose group and by day 2 in the high erythropoietin dose group. Red cell mass increased significantly on supplement day 7 in the low erythropoietin dose group and on day 5 in the high erythropoietin dose group. Fetal blood gases and pH were unchanged with time in all 3 groups.Conclusion: Although daily combined erythropoietin and iron supplements in nonanemic ovine fetuses significantly increased circulating red cell mass in a dose-dependent manner, this increase was small relative to the rapid expansion of red cell mass previously observed after iron supplementation in fetuses with hemorrhage-induced anemia. We speculate that this difference in response may be due to a combination of rapid fetal clearance of erythropoietin plus a relative insensitivity to erythropoietin caused by the absence of other cytokines, which are elevated during fetal anemia. [ABSTRACT FROM AUTHOR]

Examines the role of erythropoietin and iron in red blood cell proliferation in fetus. Elevation of circulating red cell mass level; Efficacy of erythropoietin therapy in treating anemia; Correlation of reticulocyte index and erythropoietin supplementation.

Studies the hematologic and plasma iron responses to severe fetal hemorrhage in the ovine fetus. Increase in blood volume, plasma volume, and red blood cell mass; Reticulocyte counts and plasma iron and erythropoietin levels; Posthemorrhage expansion of the red blood cell mass.

A response by Edward F. Bell, Ronald G. Strauss and John A. Widness to a letter to the editor about their article on red blood cell transfusion in neonates is presented.

Examines inter- and intraorgan iron allocation in ten twin lamb pairs undergoing an acute 40-50 percent reduction in red cell volume followed by smaller intermittent phlebotomies over an eleven-day period. Stability of tissue iron concentrations; Decrease observed in hemoprotein concentrations; What a decrease in nonheme tissue iron results in.