Because most extremely preterm infants with birth weight <1,000 g need red blood cell transfusions, many attempts have been made to collect, process and store placental blood (i.e., umbilical cord blood) for autologous transfusions. Although it is feasible to do this, multiple problems in doing so including insufficient volumes collected, clotting, hemolysis, bacterial contamination, failure to significantly supplant need for allogeneic transfusions, and high costs have led many to question whether, on balance, autologous/placental RBC transfusion offers clinically significant benefits.
placental blood; umbilical cord blood; neonatal RBC transfusions; neonatal/infant autologous RBC transfusions
To assess the long-term outcome of brain structure in preterm infants, at an average age of 12 years, who received a red blood cell transfusion for anemia of prematurity.
As neonates, this cohort of infants participated in a clinical trial in which they received red blood cell transfusions based on a high pretransfusion hematocrit threshold (liberal group) or a low hematocrit threshold (restricted group). These 2 preterm groups were compared with a group of full-term healthy control children.
Tertiary care hospital.
Magnetic resonance imaging scans for 44 of the original 100 subjects were obtained.
Liberal vs restricted transfusion.
Main Outcome Measures
Intracranial volume, total brain tissue, total cerebrospinal fluid, cerebral cortex and cerebral white matter volume, subcortical nuclei volume, and cerebellum volume.
Intracranial volume was substantially smaller in the liberal group compared with controls. Intracranial volume in the restricted group was not different from controls. Whole-cortex volume was not different in either preterm group compared with controls. Cerebral white matter was substantially reduced in both preterm groups, more so for the liberal group. The subcortical nuclei were substantially decreased in volume, equally so for both preterm groups compared with controls. When sex effects were evaluated, the girls in the liberal group had the most significant abnormalities.
Red blood cell transfusions affected the long-term outcome of premature infants as indicated by reduced brain volumes at 12 years of age for neonates who received transfusions using liberal guidelines.
Many clinical practices in transfusion medicine are controversial and/or lack definitive guidelines established by sound clinical trials. Although recommendations based on results of clinical trials performed using infants and children may not always be applied directly to adults — and vice versa — lessons learned from pediatric trials can be useful when critically assessing the design/results/conclusions of adult trials.
Four randomized clinical trials (RCTs) studying pediatric patients were critically reviewed. They addressed two RBC transfusion issues: (1) transfusion guidelines by which RBC transfusions are “triggered” by liberal (high pretransfusion patient hematocrit levels) versus being “triggered” by restricted (low pretransfusion hematocrit levels); and (2) transfusion of fresh RBCs (≤7 days storage) versus RBCs (up to 42 days storage).
Findings established by primary outcomes generally were firm (eg, fewer RBC transfusions were given to infants/children managed by restricted guidelines; transfusing small volumes of RBCs stored up to 42 days to preterm infants diminished allogeneic donor exposures and were equally efficacious and safe as fresh RBCs stored ≤7 days). Findings based on secondary outcomes, subset and post hoc analyses were inconsistent (eg, clinical outcomes were equivalent following liberal or restricted transfusions in only two of three RCTs; in the third, more neurological problems were found in neonates given restricted transfusions).
Clinical practices should be based on data pertaining to the primary outcomes of RCTs, as trials are designed and statistically powered to address these issues. Clinical practices suggested by analysis of secondary outcomes, subsets of patients and post hoc analyses, should be applied cautiously until studied further — ideally, as primary outcomes in subsequent RCTs.
Most neonates less than 1.0 kg birth weight need red blood cell (RBC) transfusions. Delayed clamping of the umbilical cord 1 minute after delivery transfuses the neonate with autologous placental blood to expand blood volume and provide 60 percent more RBCs than after immediate clamping. This study compared hematologic and clinical effects of delayed versus immediate cord clamping.
Study Design and Methods
After parental consent, neonates not more than 36 weeks' gestation were randomly assigned to cord clamping immediately or at 1 minute after delivery. The primary endpoint was an increase in RBC volume/mass, per biotin labeling, after delayed clamping. Secondary endpoints were multiple clinical and laboratory comparisons over the first 28 days including Score for Neonatal Acute Physiology (SNAP).
Problems with delayed clamping techniques prevented study of neonates of less than 30 weeks' gestation, and 105 neonates 30 to 36 weeks are reported. Circulating RBC volume/mass increased (p = 0.04) and weekly hematocrit (Hct) values were higher (p < 0.005) after delayed clamping. Higher Hct values did not lead to fewer RBC transfusions (p ≥ 0.70). Apgar scores after birth and daily SNAP scores were not significantly different (p ≥ 0.22). Requirements for mechanical ventilation with oxygen were similar. More (p = 0.03) neonates needed phototherapy after delayed clamping, but initial bilirubin levels and extent of phototherapy did not differ.
Although a 1-minute delay in cord clamping significantly increased RBC volume/mass and Hct, clinical benefits were modest. Clinically significant adverse effects were not detected. Consider a 1-minute delay in cord clamping to increase RBC volume/mass and RBC iron, for neonates 30 to 36 weeks' gestation, who do not need immediate resuscitation.
The feasibility, efficacy, and safety of transfusing stored allogeneic RBCs has been demonstrated for small-volume transfusions given to infants. We measured the posttransfusion recovery and intravascular survival of allogeneic RBCs stored up to 42 days to further elucidate their efficacy.
Study Design and Methods
Preterm infants were transfused with 1.0 mL of biotinylated RBCs plus 15 mL per kg of unlabeled allogeneic RBCs. Posttransfusion infant blood samples obtained at 10 minutes, and at 1, 2, 4, 7, 10, 14, and 21 days were used to determine the 24-hour posttransfusion recovery (PTR24), mean potential life span (MPL), and time to disappearance of 50 percent of biotinylated RBCs (T50).
No significant differences were found between allogeneic RBCs stored 1 to 21 days versus 22 to 42 days for PTR24, MPL, or T50, indicating comparable posttransfusion circulation, regardless of storage age. Although MPL and T50 values in infants using biotinylated RBCs were short, compared to those expected using chromium-labeled RBCs in adults, they agreed with results reported by others using biotinylated RBCs.
Satisfactory posttransfusion RBC recovery and survival, measured with biotinylated RBCs, support earlier clinical trials that established the efficacy and safety of stored allogeneic RBCs for small-volume transfusions given to infants. The relatively short MPL and T50 values in some infants may underestimate true survival due to ongoing erythropoiesis and infant growth with commensurate increase in blood volume during the time of RBC survival studies. Because values in infants differ from those expected using chromium-labeled RBCs in adults, and the number of posttransfusion determinations was few, additional studies are needed to define the mechanisms involved.
Many aspects of hematopoiesis are either incompletely developed in preterm infants or still functioning to serve the fetus (i.e., the intrauterine counterpart to a liveborn preterm neonate). This delayed development and/or slow adaptation to extrauterine life diminishes the capacity of the neonate to produce red blood cells (RBCs), platelets (PLTs), and neutrophils—particularly during the stress of life-threatening illnesses encountered after preterm birth such as sepsis, severe pulmonary dysfunction, necrotizing enterocolitis, and immune cytopenias. The serious medical and/or surgical problems of preterm birth can be further complicated by phlebotomy blood losses, bleeding, hemolysis, and consumptive coagulopathy. To illustrate, some preterm infants, especially those with birth weight less than 1.0 kg and respiratory distress, are given numerous RBC transfusions early in life owing to several interacting factors. Neonates delivered before 28 weeks of gestation (birth weight, <1.0 kg) are born before the bulk of iron transport has occurred from mother to fetus via the placenta and before the onset of marked erythropoietic activity of fetal marrow during the third trimester. Soon after preterm birth, severe respiratory disease can lead to repeated blood sampling for laboratory studies and, consequently, to replacement RBC transfusions. Additionally, preterm infants are unable to mount an effective erythropoietin (EPO) response to decreasing numbers of RBCs, and this factor contributes to the diminished ability to compensate for anemia—thus enhancing need for RBC transfusions.
Two randomized clinical trials, conducted independently, have reported results of neonates transfused with red blood cells (RBCs) given per either liberal (relatively high pretransfusion blood hematocrit levels) or restrictive (relatively low pretransfusion blood hematocrit levels) transfusion programs. Both found fewer RBC transfusions given per restrictive programs and comparable outcomes for several clinical endpoints. However, the Iowa trial found significantly more problems with apnea, intraparenchymal brain hemorrhage and periventricular leukomalacia in infants transfused per the restrictive program – findings not found by the Canadian trial. A critical analysis of both trials and possible reasons for the discrepant findings are discussed. Until definitive data are reported by additional studies, it seems prudent not to severely restrict/limit allogeneic RBC transfusions to neonates – except in approved investigational settings.
Neonatal RBC transfusions; RBC transfusion indications/guidelines; RBC transfusion triggers; Risks of undertransfusion, restrictive vs. liberal transfusion practices
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).
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).
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.
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 51Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for 51Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR24) for short-term RCS; 2) time to 50% decrease of the label (T50), and 3) mean potential life span (MPL) for long-term RCS.
BioRBCs tracked both normal and shortened RCS accurately relative to 51Cr. For lightly coated RBCs, mean PTR24, T50, and MPL results were not different between BioRBCs and 51Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR24 by BioRBCs (84 ± 18%) was not different from 51Cr (81 ± 10%); mean T50 by BioRBCs (23 ± 17 days) was not different from 51Cr (22 ± 18 days).
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.
The safe lower limit of hematocrit or hemoglobin that should trigger a red blood cell (RBC) transfusion has not been defined. The objective of this study was to examine the physiological effects of anemia and compare the acute responses to transfusion in preterm infants who were transfused at higher or lower hematocrit thresholds.
We studied 41 preterm infants with birth weights 500-1300 g, who were enrolled in a clinical trial comparing high (“liberal”) and low (“restrictive”) hematocrit thresholds for transfusion. Measurements were performed before and after a packed RBC transfusion of 15 ml/kg, which was administered because the infant's hematocrit had fallen below the threshold defined by study protocol. Hemoglobin, hematocrit, red blood cell count, reticulocyte count, lactic acid, and erythropoietin were measured before and after transfusion using standard methods. Cardiac output was measured by echocardiography. Oxygen consumption was determined using indirect calorimetry. Systemic oxygen transport and fractional oxygen extraction were calculated.
Systemic oxygen transport rose in both groups following transfusion. Lactic acid was lower after transfusion in both groups. Oxygen consumption did not change significantly in either group. Cardiac output and fractional oxygen extraction fell after transfusion in the low hematocrit group only.
Our results demonstrate no acute physiological benefit of transfusion in the high hematocrit group. The fall in cardiac output with transfusion in the low hematocrit group shows that these infants had increased their cardiac output to maintain adequate tissue oxygen delivery in response to anemia and, therefore, may have benefitted from transfusion.
Neonatology; haematology; circulatory; physiology; clinical procedures
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 (51Cr) -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) post-transfusion BioRBC recovery at 24 hour (PTR24); 2) time to decrease to 50% of the enrichment at 24 hours (T50); and 3) mean potential lifespan (MPL).
Among the four BioRBC densities, no significant differences in PTR24 were observed. T50 and MPL were similar for the two lowest BioRBC densities. In contrast, the two highest BioRBC densities demonstrated progressively decreased T50 and MPL.
RBCs labeled at four biotin densities can be used to independently and accurately measure PTR24 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 51Cr 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.
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 cell volume (RCV) and blood volume are 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 per 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.
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.
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.
To investigate the pathophysiology of anemia and responses to RBC transfusions and erythropoietin, repeated measurement of the circulating red 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 96 µg biotinylation reagent per mL RBC). The densities were mixed and infused intravenously. Blood was sampled five times over one hour beginning at 4 minutes. RCV values were determined based on dilution of each population of biotinylated RBCs and by the 14C-cyanate method.
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 14C-cyanate method paralleled results by the biotin method but averaged 8% greater.
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.
circulating red cell volume; blood volume; biotin; multiple densities; sheep; spleen
Most infants with birth weight <1.0 kg are given multiple red blood cell (RBC) transfusions within the first few weeks of life. The anaemia of prematurity is caused by untimely birth occuring before placental iron transport and fetal erythropoiesis are complete, by phlebotomy blood losses taken for laboratory testing, by low plasma levels of erythropoietin due to both diminished production and accelerated catabolism, by rapid body growth and need for commensurate increase in red cell volume/mass, and by disorders causing RBC losses due to bleeding and/or hemolysis. RBC transfusions are the mainstay of therapy with recombinant human erythropoietin largely unused because it fails to substantially diminish RBC transfusion needs — despite exerting substantial erythropoietic effects on neonatal marrow.
We conducted a trial of prophylactic platelet transfusions to evaluate the effect of platelet dose on bleeding in patients with hypoproliferative thrombocytopenia.
We randomly assigned hospitalized patients undergoing hematopoietic stem-cell transplantation or chemotherapy for hematologic cancers or solid tumors to receive prophylactic platelet transfusions at a low dose, a medium dose, or a high dose (1.1×1011, 2.2×1011, or 4.4×1011 platelets per square meter of body-surface area, respectively), when morning platelet counts were 10,000 per cubic millimeter or lower. Clinical signs of bleeding were assessed daily. The primary end point was bleeding of grade 2 or higher (as defined on the basis of World Health Organization criteria).
In the 1272 patients who received at least one platelet transfusion, the primary end point was observed in 71%, 69%, and 70% of the patients in the low-dose group, the medium-dose group, and the high-dose group, respectively (differences were not significant). The incidences of higher grades of bleeding, and other adverse events, were similar among the three groups. The median number of platelets transfused was significantly lower in the low-dose group (9.25×1011) than in the medium-dose group (11.25×1011) or the high-dose group (19.63×1011) (P = 0.002 for low vs. medium, P<0.001 for high vs. low and high vs. medium), but the median number of platelet transfusions given was significantly higher in the low-dose group (five, vs. three in the medium-dose and three in the high-dose group; P<0.001 for low vs. medium and low vs. high). Bleeding occurred on 25% of the study days on which morning platelet counts were 5000 per cubic millimeter or lower, as compared with 17% of study days on which platelet counts were 6000 to 80,000 per cubic millimeter (P<0.001).
Low doses of platelets administered as a prophylactic transfusion led to a decreased number of platelets transfused per patient but an increased number of transfusions given. At doses between 1.1×1011 and 4.4×1011 platelets per square meter, the number of platelets in the prophylactic transfusion had no effect on the incidence of bleeding. (ClinicalTrials.gov number, NCT00128713.)
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.
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.
Infants in the liberal-transfusion group received more RBC transfusions (5.2 ± 4.5 [mean ± SD] vs 3.3 ± 2.9 in the restrictive-transfusion group). However, the number of donors to whom the infants were exposed was not significantly different (2.8 ± 2.5 vs 2.2 ± 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.
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.
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 quantified total blood concentration of biotin label on biotin-labeled RBCs using (125I)streptavidin. The second 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.
biotin; (14C)cyanate; 51Cr; erythrocyte; red cell volume; sheep; infant
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.
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.
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.