The effect of anticoagulant, storage temperature and dilution on cord blood hematology parameters over time.

The objective of the study was to determine whether selected hematologic parameters measured on umbilical cord blood samples using an automated hematology analyzer (Sysmex XE-2100) were affected by (i) anticoagulant (the specimens were collected in EDTA vs. sodium heparin), (ii) temperature (the specimens were maintained at 4 degrees C vs. room temperature for up to 72 h) and (iii) 1 : 5 dilution vs. undiluted using the manufacturer's diluting solution. Use of heparin, instead of EDTA, had little effect on the hematologic results (n = 8) except for lower platelet and progenitor cell counts. Results were remarkably stable for 72 h at either room temperature or 4 degrees C except for modest red blood cell swelling at 24 h. Specimens of blood diluted at 1 : 5 had an immediate small, but significant change on white cell count (+13.3%), reticulocyte count (-11.2%) and reticulocyte hemoglobin content (-19.6%). Diluted samples did not change further over 4 h at room temperature. With a 1 : 5 dilution, analysis of 40 microl of cord blood stored for 3 days at room temperature may provide useful hematologic information with little phlebotomy loss.

Change in erythropoietin pharmacokinetics following hematopoietic transplantation.

Pre-clinical studies have demonstrated that bone marrow ablation has a profound effect in decreasing erythropoietin (EPO) elimination. The study's objective was to determine in humans if EPO pharmacokinetics (PKs) are perturbed following bone marrow ablation. EPO PK studies were performed in eight subjects, aged 4 to 61 years, undergoing fully myeloablative hematopoietic stem cell transplantation. Serial PK studies using intravenous injection of recombinant human EPO (92+/-2.0 U/kg) (mean+/-SEM) were carried out during four periods of altered marrow integrity: baseline pre-ablation, post-ablation pre-transplant, early post-transplant pre-engraftment, and late post-transplant full engraftment. Compared with baseline, post-ablation pre-transplant and early post-transplant EPO PKs demonstrated declines in clearance increases in terminal elimination half-life of 36 and 95%, respectively. Clearance and half-life returned to baseline following full engraftment. The association of EPO elimination with decreased bone marrow activity in patients undergoing transplantation conclusively establishes the bone marrow as a key determinant of EPO elimination in humans.

A 'bottom-up' approach for endo-PK/PD analysis.

A 'bottom-up' PK/PD analysis approach employing system analysis principles of convolution/deconvolution and special nonparametric estimation procedures is presented to resolve the complex 'endo-PK/PD' of the endogenous form of recombinant drugs using erythropoietin (EPO) as an example. A novel cellular deconvolution algorithm is presented that facilitates the identification of the functional relationship between the variables involved in EPO's complex PK/PD. Five sheep each underwent two phlebotomies spaced 4-6 weeks apart when their hemoglobin levels were reduced from 12 g/dl to 3-4 g/dl. EPO levels and reticulocyte counts were frequently sampled. The data were analysed using end-constrained cubic splines. The rate of reticulocyte production was determined using the novel deconvolution methodology. The erythroid progenitor cells activation rate by EPO was estimated from the reticulocyte production rate using a lag-time parameter which determines the delay in the reticulocyte appearance in the blood relative to the activation of erythroid progenitors. Hysteresis minimization combined with cellular deconvolution was employed to determine the population PK/PD transduction function relating the progenitor activation rate to EPO concentrations in a nonparametric manner without assuming a specific structure. The proposed approach provides a rational informative starting point for developing parametric PK/PD models to resolve the complex endo-PK/PD of recombinant drugs.

Erythropoietin production rate in phlebotomy-induced acute anemia.

OBJECTIVE: To estimate the rate of erythropoietin (EPO) production under physiological, conditions and to examine the regulatory mechanism of EPO production in response to acute phlebotomy-induced anemia. METHODS: Six sheep each underwent two phlebotomies in which the hemoglobin (Hb) was reduced to 3-4 g/dl over 4-5 h. The EPO plasma level, reticulocytes, Hb and EPO clearance were followed by frequent blood sampling. The EPO production rate was determined by a semi-parametric method based on a disposition decomposition analysis that accounts for the nonlinear disposition kinetics of EPO and corrects for time-dependent changes in the clearance. RESULTS: The controlled drop in hemoglobin resulted in an abrupt increase in the plasma EPO concentration (peak level 812+/-40 mU/ml, mean+/-CV%) that was followed by a rapid drop 2-4 days after the phlebotomy at a time when the sheep were still anemic (Hb=4.3+/-16 g/dl). The EPO production rate at baseline was 43+/-52 U/day/kg and the amounts of EPO produced over an 8 day period resulting from the first and second phlebotomy were 2927+/-40 U/kg and 3012+/-31 U/kg, respectively. CONCLUSIONS: The rapid reduction in the EPO plasma level observed 2-4 days following the phlebotomy cannot be explained solely by the increase in EPO clearance but also by a reduction in EPO production.

Pharmacokinetic tracer kinetics analysis of changes in erythropoietin receptor population in phlebotomy-induced anemia and bone marrow ablation.

OBJECTIVES: The objective was to study in vivo erythropoietin (Epo) progenitor cell surface receptors (EpoR) in the bone marrow (BM) after phlebotomy and bone marrow ablation. METHODS: Serial tracer interaction method experiments were conducted in adult sheep at baseline and after phlebotomy (PH) and ablation (AB). PH was done 10 days after phlebotomy (to 3-4 g/dl Hb), and the AB was done 8 days after a 3-day oral treatment with bulsulfan (11 mg/kg/day). RESULTS: Bone marrow ablation changed the elimination from non-linear to linear, consistent with an abolition of the non-linear elimination via BM EpoRs. The phlebotomy increased the linear clearance of the ablated elimination pathway (from 63.6+/-12 to 126+/-64 ml/h/kg), consistent with an up-regulation of the erythroid progenitor BM-based EpoR pool, but did not change the clearance of the non-ablated elimination pathway (p>0.05). The EpoR pool size remaining after BM ablation was 7.4+/-2.7% of the pre-ablation pool. CONCLUSIONS: Erythropoietin elimination via EpoR in the bone marrow was non-linear and increased following phlebotomy-induced anemia. This is consistent with an up-regulation of the erythropoietic EpoR pool in BM. Assuming that the elimination of Epo after BM ablation was via non-hematopoietic EpoR, then this post-ablation EpoR population was not significantly up-regulated by the phlebotomy.

Pharmacokinetic/pharmacodynamic analysis of paradoxal regulation of erythropoietin production in acute anemia.

The regulatory mechanism responsible for a paradoxal, rapid drop in the erythropoietin (EPO) plasma level seen 2 to 4 days after acute, phlebotomy-induced anemia was investigated in seven adult sheep. To introduce acute anemia, each sheep underwent two phlebotomies where the hemoglobin (Hb) was reduced to 3 or 4 g/dl over 4 to 5 h. The phlebotomies were spaced 4 to 6 weeks apart in three animals, and 8 days apart in four other animals. EPO plasma levels, reticulocyte count, Hb, and p50 for oxygen-Hb dissociation were determined from frequent blood samplings throughout the study period. EPO's disposition pharmacokinetic (PK) and plasma clearance were determined from i.v. bolus injections of tracer amounts of a recombinant human EPO tracer. The controlled drop in Hb resulted in a rapid increase in plasma EPO to 836 +/- 52 mU/ml (mean +/- coefficient of variation percentage) that was followed by a paradoxical rapid drop 2 to 4 days after the phlebotomy while the animals were still very anemic (Hb = 4.3 +/- 15 g/dl). The rapid drop in plasma EPO level could not be explained by the up-regulated clearance (clearance increased by a factor of less than 2.5) or by physiological adaptation (no change in p50, p > 0.05, second phlebotomy to Hb = 3g/dl inadequately stimulated the EPO production). The PK/pharmacodynamic (PD) analysis supports the hypothesis of a limited sustained high EPO production rate in acute anemia, which indicates an apparent deficiency in the regulation of EPO production in acute anemia. The hypothesis was supported by a PK/PD feedback inhibition model that showed good agreement with the data (r = 0.973 +/- 1.57).

A differential pharmacokinetic analysis of the erythropoietin receptor population in newborn and adult sheep.

Strong evidence indicates that erythropoietin (Epo) is eliminated via Epo receptors (EpoR). Epo receptors may be classified as erythropoietic receptors that are largely located on erythroid progenitor cells in the bone marrow (BM) and nonerythropoietic receptors present in most tissues. Epo's elimination kinetics was studied using a very sensitive tracer interaction method (TIM) before and after chemical ablation of BM as an indirect way of evaluating the EpoR through an assortment of pharmacokinetic parameters (VM, KM, K, and CL) used in differentiating the EpoR population in newborn and adult sheep. TIM identified a parallel nonlinear Michaelis-Menten (VM and KM), and linear (K) elimination pathway and found the latter pathway to be significantly (p < 0.01) more dominant in lamb: K/(VM/KM + K) = 0.309 (25.3) versus 0.0895 (18.4) mean (CV%) lambs versus adult sheep. The significantly (p < 0.01) larger total clearance found for lambs indicates a larger nonhematopoietic tissue clearance of Epo (CL = 118 (10.9) ml/h/kg versus 67.8 (19.3) lamb versus adult sheep). The VM/KM ratio for the nonlinear pathway was not found to be significantly different (p > 0.05) between newborn and adults with values of 1.10 (15.8) and 1.30 (3.81) h-1, respectively. We proposed the hypothesis that the linear pathway is via nonhematopoietic EpoR. Assuming that Epo's elimination largely depends not only on erythropoietic EpoR but also on nonhematopoietic EpoR, this work shows a significant difference in the relative proportions of the two EpoR populations in lamb and adult sheep. The larger dominance of the nonhematopoietic EpoR in lamb supports the hypothesis that these receptors are more needed in early life, e.g., providing neuroprotection from perinatal hypoxemic-ischemic episodes.

An integrated pharmacodynamic analysis of erythropoietin, reticulocyte, and hemoglobin responses in acute anemia.

PURPOSE: To determine by pharmacodynamic (PD) analysis physiologically relevant parameters of the cellular kinetics of erythropoiesis in acute anemia. METHODS: The PD relationships among erythropoietin (EPO), reticulocyte, and RBC (Hb) responses were investigated in young adult sheep in acute anemia induced twice by two controlled phlebotomies separated by a 4-week recovery period. RESULTS: The phlebotomies resulted in rapid increases in plasma EPO, with maximal levels occurring at 3 to 8 days, followed by a reticulocyte response with a delay of 0.5 to 1.5 days. The Hb returned to prephlebotomy base line at the end of the 4-week recovery period. The EPO, reticulocyte count, and Hb responses were well described by a PK/PD model (r = 0.975) with the following cellular kinetics parameters: the lag time between EPO activation of erythroid progenitor cells and reticulocyte formation; the reticulocyte-to-RBC maturation time; the reticulocyte and Hb formation efficacy coefficients, quantifying EPO's efficacy in stimulating the formation of reticulocytes and Hb, respectively; the C50 PK/PD transduction parameter defined as the EPO level resulting in half the maximum rate of erythropoiesis. CONCLUSION: Physiologically relevant cellular kinetics parameters can be obtained by an endogenous PK/PD analysis of phlebotomy data and are useful for elucidating the pathophysiologic etiology of various anemias.

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.

Erythropoietin responses to progressive blood loss over 10 days in the ovine fetus.

Long-term loss of fetal blood can occur with fetomaternal hemorrhage, vasoprevia, or placental previa. Our objective was to determine the effects of progressive fetal blood loss over 10 days on fetal plasma erythropoietin (EPO) concentration and its relationship to arterial PO(2), hematocrit, and the volume of blood loss. Late-gestation fetal sheep (n = 8) were hemorrhaged daily at a rate of 1 ml/min over 10 days. The extent of hemorrhage differed in each fetus and ranged from 30 to 80 ml/day, with the cumulative volume removed ranging from 78 to 236 ml/kg estimated fetal weight. Four fetuses served as time controls. EPO concentration measurements were by radioimmunoassay. Statistical analyses included regression, correlation, and analysis of variance. We found that EPO and arterial PO(2) were unchanged until the cumulative hemorrhage volume exceeded 20-40 ml/kg. Once this threshold was exceeded, plasma EPO concentration increased progressively throughout the study and averaged 14.3 +/- 3.2 times basal values on day 10. EPO concentration, arterial PO(2), and hematocrit changes were related curvilinearly to cumulative hemorrhage volume (P < 0.01), whereas the relationship between plasma EPO and arterial PO(2) was log linear (P < 0.001). We conclude that 1) fetal plasma EPO concentration and arterial PO(2) are insensitive to a slow, mild-to-moderate blood loss over several days; 2) unlike the rapid return of EPO to normal within 48 h after acute hemorrhage, fetal EPO concentration undergoes a progressive increase with moderate-to-severe blood loss over several days; 3) the long-term hemorrhage-induced changes in EPO are best correlated with arterial PO(2); and 4) the fetal EPO response to hemorrhage does not appear to be limited by the fetus's ability to produce EPO.

Changes in erythropoietin pharmacokinetics following busulfan-induced bone marrow ablation in sheep: evidence for bone marrow as a major erythropoietin elimination pathway.

The contribution of the bone marrow to in vivo erythropoietin (EPO) elimination was evaluated by determining EPO pharmacokinetic (PK) parameters in five adult sheep in a paired manner before and after chemotherapy-induced marrow ablation. After busulfan-induced bone marrow ablation, EPO PK demonstrated progressive decreases in plasma clearance (CL), elimination half-life [t1/2(beta)], and volume of distribution at steady state (Vss) with concomitant increases in mean residence time (MRT). Eight days after beginning busulfan treatment, there were no further changes in CL, t1/2(beta), MRT, and Vss. Only 20% of baseline CL remained by day 8. The volume of distribution (Vc) and distribution half-life [t1/2(alpha)], in contrast, remained unchanged from baseline. White blood cell counts and reticulocytes gradually declined after the start of marrow ablation. Examination of bone marrow core biopsy samples obtained on day 10 revealed less than 10% of baseline marrow cellularity. No colony-forming unit erythroid (CFU-E) colonies were found after 6 days of incubation for bone marrow aspirates drawn at days 8 and 13 following busulfan treatment, whereas pre-busulfan aspirates yielded 29 CFU-E colonies per 10(5) cells in CFU-E cultures. Treatment of a sheep with 5-fluorouracil showed changes in PK parameters that were similar to the results from treatment with busulfan. The present study indicates that the bone marrow significantly contributes to the elimination of EPO in vivo.

Expanded Möbius syndrome.

A woman presented at 33 weeks gestation with reduced fetal movements and a nonreactive nonstress test. Fetal ultrasound examination revealed a peculiar unilateral arm tremor. At emergency cesarean section, performed for fetal indications, a 1,672-gm male infant was delivered requiring intubation for feeble respiratory effort. After delivery the neonate was transiently hypertonic and later hypotonic. Continuing ventilatory support at minimal settings was necessary. The work-up for aneuploidy, metabolic disorders, and infection was negative. The infant died after being removed from ventilatory support on day 22. Postmortem examination revealed extensive bilateral brain gliosis and mineralization without evidence of inflammation, partial absence of cranial nerve nuclei III-XI, and a total absence of cranial nerve roots VI-XI. Together these finding are compatible with a diagnosis of expanded Möbius syndrome.

Etiology of differences in hematocrit between males and females: sequence-based polymorphisms in erythropoietin and its receptor.

INTRODUCTION: Males have a higher hematocrit (Hct) than females. The cause of this gender-based difference is unclear. We sought to determine whether polymorphisms of the erythropoietin (EPO) gene or of its receptor (EPOR) explain this situation. METHODS: We designed primers for the EPO and EPOR genes. Previously undescribed polymorphisms were found based on band migration on polyacrylamide gel, and when then sequenced. The distribution of these polymorphisms was studied in a population of 819 non-iron-deficient, healthy blood donors. To test the gender differences, analysis was done based on groups defined by Hct levels. The chi-square statistic was used to compare the frequency differences between groups, with P < .05 considered statistically significant. RESULTS: We found previously reported polymorphisms in both the EPO and EPOR genes. Sequence analysis showed that the EPO polymorphism was due to a difference in the repeat copy number of the tetranucleotide cytosine adenine cytosine thymine (CACT) at position 2153. A previously undescribed 12th allele was found for the EPOR polymorphic site. Statistical analysis showed that the EPOR alleles, EPORA1 and EPORA10, were present at a significantly higher frequency in females than in males (P = .027 and P = .041, respectively), and EPOR5 was found less frequently in females than in males (P = .048). The allelic frequency of the EPO polymorphism was not significantly different by gender or Hct groups. DISCUSSION: These results suggest that the variation of Hct level by gender may have a genetic basis. The sequence-based polymorphism for EPOR may be partly responsible for this gender-based variation in Hct level. These findings offer new clues to understanding Hct variation in the general population and to elucidating mechanisms of controlling Hct levels.

Receptor-based model accounts for phlebotomy-induced changes in erythropoietin pharmacokinetics.

Previous clinical studies have demonstrated two distinctive pharmacokinetic behaviors of erythropoietin (EPO): changes in pharmacokinetics (PK) after a period of rhEPO treatment and nonlinear pharmacokinetics. The objective of this work was to study the temporal changes in EPO's PK following phlebotomy in order to propose possible mechanisms for this behavior. Five healthy adult sheep were phlebotomized on two separate occasions 4-6 weeks apart to hemoglobin levels of PK 3-4 g/dL. PK parameters were estimated from the concentration-time profiles obtained following repeated intravenous bolus PK studies using tracer doses of biologically active 125I-rhEPO. Based on the changes in clearances, a PK model was derived to provide a mechanistic receptor-based description of the observed phenomena. Phlebotomy resulted in a rapid increase in the EPO plasma concentration, which peaked at 760 +/- 430 mU/mL (mean +/- SD) at 1.8 +/- 0.65 days, and which coincided with a transient reduction in EPO clearance from prephlebotomy values, i.e., from 45.6 +/- 11.2 mL/hr/kg to 24.3 +/- 9.7 mL/hr/kg. As plasma EPO levels returned toward baseline levels in the next few days, a subsequent increase in EPO clearance was noted. EPO clearance peaked at 90.2 +/- 26.2 mL/hr/kg at 8.5 +/- 3.3 days and returned to baseline by 4-5 weeks postphlebotomy. The proposed model derived from these data includes positive feedback control of the EPO receptor (EPOR) pool. The model predicts that: 1) the initial reduction in EPO plasma clearance is due to a transient saturation of EPORs resulting from the phlebotomy-induced high EPO concentration; and 2) the EPOR pool is expandable not only to compensate for EPOR loss but also to adjust to a greater need for EPORs/progenitor cells to restore hemoglobin (Hb) concentration to normal levels.

Effect of intravenous iron supplementation on erythropoiesis in erythropoietin-treated premature infants.

OBJECTIVE: To test the efficacy and safety of combining intravenous iron in amounts approximating the in utero iron accretion rate and the postnatal iron loss with erythropoietin (EPO) in very low birth weight (VLBW) infants. METHODS: A prospective, controlled, randomized, unmasked trial lasting 21 days was performed in 29 clinically stable VLBW infants <31 weeks' gestation and <1300 g birth weight not treated with red blood cell transfusions during the study period. Mean (+/- standard error of the mean) age at study entry was 23 +/- 2.9 days. After a 3-day run-in baseline period in which all participants received oral supplements of 9 mg/kg/day of iron polymaltose complex (IPC), participants were randomized to receive 18 days of treatment with: 1) oral IPC alone (oral iron group); 2) 300 U of recombinant human EPO (r-HuEPO) kg/day and daily oral IPC (EPO + oral iron group); 3) 2 mg/kg/day of intravenous iron sucrose, r-HuEPO, and oral iron (intravenous iron + EPO group). To assess efficacy of the 3 treatments, serial blood samples were analyzed for hemoglobin (Hb), hematocrit (Hct), reticulocyte count, red blood cell indices and plasma levels of transferrin, transferrin receptor (TfR), ferritin, and iron. Oxidant injury was assessed before and after treatment by plasma and urine levels of malondialdehyde (MDA) and o-tyrosine. RESULTS: At the end of treatment, Hb, Hct, reticulocyte count, and plasma TfR were markedly higher in both of the EPO-treated groups, compared with the oral iron group. At study exit a trend toward increasing Hb and Hct levels and significantly higher reticulocyte counts were observed in the intravenous iron + EPO group, compared with the EPO + oral iron group. During treatment, plasma ferritin levels increased significantly in the intravenous iron + EPO group and decreased significantly in the other 2 groups. By the end of treatment, ferritin levels were significantly higher in the intravenous iron + EPO group compared with the other 2 groups. Although plasma and urine MDA or o-tyrosine did not differ among the 3 groups, plasma MDA was significantly greater in the subgroup of intravenous iron + EPO participants sampled at the end of the 2-hour parenteral iron infusion, compared with values observed immediately before and after parenteral iron-dosing. CONCLUSIONS: In stable VLBW infants receiving EPO treatment, parenteral supplementation with 2 mg/kg/day of iron sucrose results in a small, but significant, augmentation of erythropoiesis beyond that of r-HuEPO and enteral iron alone. However, to reduce the potential adverse effects of parenteral iron/kg/day on increasing plasma ferritin levels and on causing oxidative injury, we suggest that the parenteral iron dose used should be reduced and/or the time of infusion extended to maintain a serum iron concentration below the total iron-binding capacity.

A pharmacodynamic analysis of erythropoietin-stimulated reticulocyte response in phlebotomized sheep.

The pharmacodynamics (PD) of the reticulocyte response resulting from phlebotomy-induced erythropoietin (EPO) was investigated in adult sheep. The anemia caused by the controlled phlebotomy (Hb < 4 g/dl, t = 0) resulted in a rapid increase in EPO with peak concentrations from 200 to 1400 mU/ml at 0.5 to 3 days generating a delayed reticulocyte response with peak levels from 9.3 to 14.1% at 2.5 to 5.1 days. The PD EPO-reticulocyte relationship is well described by a simple kinetic model involving 3 relevant physiologic parameters: T(1) = lag-time (0.73 +/- 0.32 days, mean +/- S.D.), T(2) = reticulocyte maturation time (5.61 +/- 1.41 days), and k = EPO efficacy coefficient (0.052 +/- 0.048% g/dl mU/ml/day). Accordingly, 0.52% reticulocytes at 10 g/dl Hb level are generated per day at an EPO concentration of 100 mU/ml. The difference between the T(2) parameter in this study and the maturation time reported for humans may be due to interspecies differences or different technique and experimental conditions. The PD transduction appears largely linear in the observed EPO concentration range, indicating a full utilization of EPO without any significant PD saturation. Also, the EPO concentration versus time profiles resulting from the phlebotomy were similar to exogenous EPO profiles resulting from s.c. therapeutic dosing. This study supports the hypothesis that s.c. EPO dosing is more efficacious than i.v. dosing.

Clinical performance of an in-line point-of-care monitor in neonates.

OBJECTIVE: To evaluate the bias, precision, and blood loss characteristics of an ex vivo in-line point-of-care testing blood gas and electrolyte monitor designed for use in critically ill newborn infants. STUDY DESIGN: Study participants included consecutive neonates with an umbilical artery catheter (UAC) in use for clinical laboratory testing. The in-line monitor (VIA LVM Blood Gas and Chemistry Monitoring System, VIA Medical, San Diego, CA) was directly connected to the participant's UAC and the monitor's determinations of pH, PCO(2), PO(2), sodium, potassium, and hematocrit (Hct) were compared with those simultaneously drawn and measured with a standard bench top laboratory instrument (Radiometer 625 ABL; Radiometer America, Inc, Westlake, OH). The bias (the mean difference from the reference method) and precision (1 standard deviation of the mean difference) performance criteria of the in-line monitor were derived using standard laboratory procedures. RESULTS: Sixteen neonates monitored for a total of 37 days had a total of 229 paired blood samples available for comparison by the 2 methods. Bias and precision performance characteristics of the in-line monitor were similar to reports of other point-of-care devices (ie, pH: -.003 +/-.024; PCO(2):.35 +/- 2.84 mm Hg; PO(2):.39 +/- 7.30 mm Hg; sodium:.52 +/- 2.34 mmol/L; potassium:.17 +/-.18 mmol/L; and Hct:.61 +/- 2.80%). The range of values observed for each parameter included much of the range anticipated among critically ill neonates (ie, pH: 7.15-7.65; PCO(2): 25-75 mm Hg; PO(2): 25-275 mm Hg; sodium: 127-150 mmol/L; potassium: 2.6-5.5 mmol/L; and Hct: 32%-60%). Mean blood loss (+/- standard deviation) per sample with the in-line monitor was approximately one-tenth that of the reference method: 24 +/- 7 microL versus 250 microL, respectively. There was no evidence of hemolysis and no patient related safety issues were identified with use of the in-line monitor. CONCLUSIONS: Repeated laboratory testing of critically ill neonates using an ex vivo in-line monitor designed for use in neonates provides reliable laboratory results. The blood loss and hemolysis data obtained suggests that this monitoring device offers potential for reducing neonatal blood loss-and possibly transfusion needs-during the first weeks of life. Before this promising technology can be routinely recommended for care of critically ill neonates, greater practical experience in a variety of clinical settings is needed.

Phlebotomy overdraw in the neonatal intensive care nursery.

OBJECTIVE: Because blood loss attributable to laboratory testing is the primary cause of anemia among preterm infants during the first weeks of life, we quantified blood lost attributable to phlebotomy overdraw, ie, excess that might be avoided. We hypothesized that phlebotomy overdraw in excess of that requested by the hospital laboratory was a common occurrence, that clinical factors associated with excessive phlebotomy loss would be identified, and that some of these factors are potentially correctable. DESIGN, OUTCOME MEASURES, AND ANALYSIS: Blood samples drawn for clinical purposes from neonates cared for in our 2 neonatal special care units were weighed, and selected clinical data were recorded. The latter included the test performed; the blood collection container used; the infant's location (ie, neonatal intensive care unit [NICU] and intermediate intensive care unit); the infant's weight at sampling; and the phlebotomist's level of experience, work shift, and clinical role. Data were analyzed by univariate and multivariate procedures. Phlebotomists included laboratory technicians stationed in the neonatal satellite laboratory, phlebotomists assigned to the hospital's central laboratory, and neonatal staff nurses. Phlebotomists were considered experienced if they had worked in the nursery setting for >1 year. Blood was sampled from a venous or arterial catheter or by capillary stick from a finger or heel. Blood collection containers were classified as tubes with marked fill-lines imprinted on the outside wall, tubes without fill-lines, and syringes. Infants were classified by weight into 3 groups: <1 kg, 1 to 2 kg, and >2 kg. The volume of blood removed was calculated by subtracting the weight of the empty collection container from that of the container filled with blood and dividing by the specific gravity of blood, ie, 1.050 g/mL. The volume of blood withdrawn for individual laboratory tests was expressed as a percentage of the volume requested by the hospital laboratory. RESULTS: The mean (+/- standard error of the mean) volume of blood drawn for the 578 tests drawn exceeded that requested by the hospital laboratory by 19.0% +/- 1.8% per test. The clinical factors identified as being significantly associated with greater phlebotomy overdraw in the multiple regression model included: 1) collection in blood containers without fill-lines; 2) lighter weight infants; and 3) critically ill infants being cared for in the NICU. Because the overall R(2) of the multiple regression for these 3 clinical factors was only.24, the random factor of individual phlebotomist was added to the model. This model showed that there was a significant variation in blood overdraw among individual phlebotomists, and as a result, the overall R(2) increased to.52. An additional subset analysis involving 2 of the 3 groups of blood drawers (ie, hospital and neonatal laboratory phlebotomists) examining the effect of work shift, demonstrated that there was significantly greater overdraw for blood samples obtained during the evening shift, compared with the day shift when drawn using unmarked tubes for the group of heavier infants cared for in the NICU. CONCLUSION: Significant volumes of blood loss are attributable to overdraw for laboratory testing. This occurrence likely exacerbates the anemia of prematurity and may increase the need for transfusions in some infants. Attempts should be made to correct the factors involved. Common sense suggests that blood samples drawn in tubes with fill-lines marked on the outside would more closely approximate the volumes requested than those without. Conversely, the use of unmarked tubes could lead to phlebotomy overdraw because phlebotomists may overcompensate to avoid having to redraw the sample because of an insufficient volume for analysis. We were surprised to observe that the lightest and most critically ill infants experienced the greatest blood overdraw. (ABSTRACT TRUNCATED)

Association of fetal and maternal carboxyhemoglobin levels in normal and Rh-alloimmune pregnancies.

OBJECTIVE: To compare paired antepartum fetal/maternal COHb ratios in whole blood from control and alloimmunized pregnancies and to examine the relationships between fetal and maternal COHb. METHODS: COHb levels were measured in paired fetal and maternal blood samples obtained at cordocentesis in 47 control and 16 Rh-alloimmunized pregnancies. COHb was determined by gas chromatography. Results were analyzed by t-test, regression and analysis of covariance. RESULTS: Although fetal/maternal COHb ratios for control and alloimmunized pregnancies were not statistically significantly different, i.e. 1. 11+/-0.04 and 1.26+/-0.09, respectively (P=0.09), fetal COHb levels were higher in Rh-alloimmunized fetuses (P=0.0002). Fetal COHb levels were also higher than paired maternal levels among the alloimmunized group (P=0.011), but not among the control group (1. 04+/-0.04, P=ns). In univariate regression analysis, fetal and maternal COHb levels were significantly correlated with one another in both control (r=0.52, P=0.0002) and alloimmunized pregnancy groups (r=0.52, P=0.05). Comparison of the slopes of the fetal versus maternal COHb plots for the two groups showed a significant difference (P=0.02), with the alloimmunized group having the steeper slope. CONCLUSION: Differences in the antepartum fetal-maternal COHb relationships in control and alloimmunized groups likely reflect increased endogenous CO production among alloimmunized fetuses as a result of pathologic hemolysis.

Adaptive responses during anemia and its correction in lambs.

There is limited information available on which to base decisions regarding red blood cell (RBC) transfusion treatment in anemic newborn infants. Using a conscious newborn lamb model of progressive anemia, we sought to identify accessible metabolic and cardiovascular measures of hypoxia that might provide guidance in the management of anemic infants. We hypothesized that severe phlebotomy-induced isovolemic anemia and its reversal after RBC transfusion result in a defined pattern of adaptive responses. Anemia was produced over 2 days by serial phlebotomy (with plasma replacement) to Hb levels of 30-40 g/l. During the ensuing 2 days, Hb was restored to pretransfusion baseline levels by repeated RBC transfusion. Area-under-the-curve methodology was utilized for defining the Hb level at which individual study variables demonstrated significant change. Significant reciprocal changes (P < 0.05) of equivalent magnitude were observed during the phlebotomy and transfusion phases for cardiac output, plasma erythropoietin (Epo) concentration, oxygen extraction ratio, oxygen delivery, venous oxygen saturation, and blood lactate concentration. No significant change was observed in resting oxygen consumption. Cardiac output and plasma Epo concentration increased at Hb levels <75 g/l, oxygen delivery and oxygen extraction ratio decreased at Hb levels <60 g/l, and venous oxygen saturation decreased and blood lactate concentration increased at Hb levels <55 g/l. We speculate that plasma Epo and blood lactate concentrations may be useful measures of clinically significant anemia in infants and may indicate when an infant might benefit from a RBC transfusion.