Demet Nalbant - Academia.edu (original) (raw)
Papers by Demet Nalbant
Transfusion, May 16, 2018
The current reference method in the U.S.A. for measuring in vivo population red blood cell (RBC) ... more The current reference method in the U.S.A. for measuring in vivo population red blood cell (RBC) kinetics utilizes 51-chromium (51 Cr) RBC labeling for determining red cell volume (RCV), 24hour post-transfusion RBC recovery (PTR 24), and long-term red cell survival (RCS). Here we provide evidence supporting adoption of a method for kinetics that uses the biotin labeled RBC *
Transfusion, Jan 16, 2018
The current reference method in the United States for measuring in vivo population red blood cell... more The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-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 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 pharm...
Antimicrobial agents and chemotherapy, Jan 25, 2018
The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a s... more The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of their plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous determination of multiple β-lactam antibiotics and β-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin and tazobactam in human plasma was developed and rigorously validated according to the FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using a step-wise gradient elution. Analytes were monitored using MS/MS with a turbo ion spray source in positive ...
Transfusion, Jan 29, 2017
Critically ill preterm very-low-birthweight (VLBW) neonates (birthweight ≤ 1.5 kg) frequently dev... more 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 (PTR24 ) 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 PTR24 with storage. We hypothesized that PTR24 of allogeneic RBCs transfused to anemic VLBW newborns: 1) will be greater than PTR24 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. PTR24 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 PTR24 versus duration of RBC storage. For VLBW newborns, the ...
Transfusion, Jun 5, 2017
Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dos... more 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. 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. 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...
Transfusion, 2014
The quality of transfused red blood cells (RBCs) to treat anemia depends on its potential for oxy... more 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. 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. 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. MRL overcomes limitations of previously proposed TRCS parameters, is simpler to calculate, and is physiologically and clinically more appropriate.
AJP: Regulatory, Integrative and Comparative Physiology, 2013
A population pharmacokinetics/pharmacodynamic (PK/PD) model was developed to describe changes in ... more A population pharmacokinetics/pharmacodynamic (PK/PD) model was developed to describe changes in erythropoiesis as a function of plasma erythropoietin (EPO) concentration over the first 30 days of life in preterm infants who developed severe anemia requiring red blood cell (RBC) transfusion. Several covariates were tested as possible factors influencing the responsiveness to EPO. Discarded blood samples in 27 ventilated preterm infants born at 24-29 wk of gestation were used to construct plasma EPO, hemoglobin (Hb), and RBC concentration-time profiles. The amount of Hb removed for laboratory testing and that transfused throughout the study period were recorded. A population PK/PD model accounting for the dynamic Hb changes experienced by these infants was simultaneously fitted to plasma EPO, Hb, and RBC concentrations. A covariate analysis suggested that the erythropoietic efficacy of EPO is increased for preterm infants at later gestational ages. The PD analysis showed a sevenfold difference in maximum Hb production rate dependent on gestational age and indicated that preterm infants, when stimulated by EPO, have the capacity to produce additional Hb that may result in a decrease in RBC transfusions. The present model has utility in clinical trial simulations investigating the treatment potential of erythropoietic stimulating agents in the treatment of anemia of prematurity.
Pediatric Research, 2017
Prior conclusions that autologous neonatal red blood cells (RBC) have substantially shorter lifes... more Prior conclusions that autologous neonatal red blood cells (RBC) have substantially shorter lifespans than allogeneic adult RBCs were not based on direct comparison of autologous neonatal vs. allogeneic adult RBCs performed concurrently in the same infant. Biotin labeling of autologous neonatal RBCs and allogeneic adult donor RBCs permits concurrent direct comparison of autologous vs. allogeneic RBC lifespan. RBCs from 15 allogeneic adult donors and from 15 very low birth weight (VLBW) neonates were labeled at separate biotin densities and transfused simultaneously into the 15 neonates. Two mathematical models that account for the RBC differences were employed to estimate lifespans for the two RBC populations. Mean±SD lifespan for adult allogeneic RBC was 70.1±19.1 d, which is substantially shorter than the 120 d lifespan of both autologous and adult allogeneic RBC in healthy adults. Mean±SD lifespan for neonatal RBC was 54.2±11.3 d, which is only about 30% shorter than that of the adult allogeneic RBCs. This study provides evidence that extrinsic environmental factors primarily determine RBC survival (e.g., small bore of the capillaries of neonates, rate of oxygenation/deoxygenation cycles) rather than factors intrinsic to RBC.Pediatric Research (2017); doi:10.1038/pr.2017.14.
Endocrinology, 1998
Leydig cells are located in the interstitium of the testis and function as the primary site for t... more Leydig cells are located in the interstitium of the testis and function as the primary site for testosterone biosynthesis. Leydig cell development and steroidogenic function are dependent upon pituitary-derived LH. Circulating LH levels in prepubertal mammals are low but rise sharply during puberty, inducing terminal differentiation of immature Leydig cells into adult Leydig cells. The molecular mechanisms involved in LH action on differentiation specific gene expression and initiation of steroidogenic function in immature Leydig cells are poorly understood. Members of the CCAAT/enhancer-binding protein (C/EBP) family of basic region/leucine zipper transcription factors have previously been implicated as regulators of terminal differentiation in several cell types. In the present study we have investigated the possible involvement of C/EBP proteins in regulating LH-dependent gene expression in Leydig cells. We have detected the expression of one family member, C/EBPbeta, in Leydig cells. C/EBPbeta messenger RNA and protein levels were significantly higher in mature adult Leydig cells than in immature cells, displaying an expression pattern similar to those of other developmentally regulated genes in Leydig cells such as steroidogenesis acute regulatory (StAR) protein and 3beta-hydroxysteroid dehydrogenase. C/EBPbeta messenger RNA and protein levels also increased when immature Leydig cells were treated with either hCG, a functional analog of LH (hCG/LH), or (Bu)2cAMP. To confirm that hCG/LH and (Bu)2cAMP were acting specifically on Leydig cells, we studied their effects on C/EBPbeta expression in an established Leydig cell line (MA-10). hCG and (Bu)2cAMP treatment also induced the expression of C/EBPbeta and StAR in MA-10 cells, coincident with stimulation of steroid production in these cells. (Bu)2cAMP treatment did not alter the subcellular localization of C/EBPbeta protein in MA-10 cells, suggesting that the increase is due to stimulation of C/EBPbeta expression. We conclude that expression of C/EBPbeta is regulated by hCG/LH in Leydig cells and that C/EBPbeta may play a significant role in LH-regulated Leydig cell differentiation and function.
The AAPS journal, Jan 26, 2015
Postnatal hemoglobin (Hb) production in anemic preterm infants is determined by several factors i... more Postnatal hemoglobin (Hb) production in anemic preterm infants is determined by several factors including the endogenous erythropoietin levels, allogeneic RBC transfusions administered to treat anemia, and developmental age. As a result, their postnatal Hb production rate can vary considerably. This work introduces a novel Hb mass balance-based semiparametric approach that utilizes infant blood concentrations of Hb from the first 30 postnatal days to estimate the amount of Hb produced and the erythropoiesis rate in newborn infants. The proposed method has the advantage of not relying on specific structural pharmacodynamic model assumptions to describe the Hb production, but instead utilizes simple mass balance principles and nonparametric regression analysis. The developed method was applied to the Hb data from 79 critically ill anemic very low birth weight preterm infants to evaluate the dynamic changes in erythropoiesis during the first month of life and to determine the inter-sub...
The Journal of pediatrics, Jan 9, 2015
Based on the hypothesis that neonatal autologous red blood cell (RBC) survival (RCS) is substanti... more Based on the hypothesis that neonatal autologous red blood cell (RBC) survival (RCS) is substantially shorter than adult RBC, we concurrently tracked the survival of transfused biotin-labeled autologous neonatal and allogeneic adult RBC into ventilated, very low birth weight infants. RBC aliquots from the first clinically ordered, allogeneic adult RBC transfusion and from autologous infant blood were labeled at separate biotin densities (biotin-labeled RBC [BioRBC]) and transfused. Survival of these BioRBCs populations were concurrently followed over weeks by flow cytometric enumeration using leftover blood. Relative tracking of infant autologous and adult allogeneic BioRBC was analyzed by linear mixed modeling of batched weekly data. When possible, Kidd antigen (Jka and Jkb) mismatches between infant and donor RBCs were also used to track these 2 populations. Contrary to our hypothesis, concurrent tracking curves of RCS of neonatal and adult BioRBC in 15 study infants did not diffe...
The AAPS journal, Jan 28, 2015
Fetal RBCs are produced during a period of very rapid growth and stimulated erythropoiesis under ... more 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 erythro...
Journal of Clinical Laboratory Analysis, 2015
Reticulocyte hemoglobin content (RET-He)-an established indicator of iron status in children and ... more Reticulocyte hemoglobin content (RET-He)-an established indicator of iron status in children and adults-was determined in very low birth weight (VLBW) infants. Longitudinal retrospective RET-He data in 26 VLBW neonates during the first month of age were compared with: (a) concurrent complete blood counts (CBCs), including hemoglobin (Hb) concentration, reticulocyte count, and immature reticulocyte fraction (IRF), and erythropoietin (EPO) levels; (b) clinical variables; and (c) RET-He data from the literature for term infants, children, and adults. RET-He within 24 hr following birth was 31.8 ± 1.1 pg (mean ± SEM). This was followed by an abrupt, significant decline to 28.3 ± 1.1 pg at 2-4 days, and to steady state levels of 28.4 ± 0.5 pg thereafter. The changes in RET-He were mirrored by changes in plasma EPO, reticulocyte count, and IRF, but not Hb. Steady state RET-He values after 4 days were significantly lower than RET-He values for term infants, children, and adults (31.6 ± 0.11, 32.0 ± 0.12, and 33.0 ± 0.13 pg, respectively). Although RET-He values in VLBW infant were lower than term infants, children, and adults, the significance and mechanism(s) responsible are unknown. The present VLBW infant data are relevant to investigations assessing hemoglobinization following treatment with recombinant human EPO (r-HuEPO) and/or iron.
Pediatric Research, 2013
Anemia, a common condition among critically ill premature infants, is affected by red blood cell ... more 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). 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. 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. 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.
BMC genomics, Jan 27, 2005
Hematopoiesis is a complex developmental process controlled by a large number of factors that reg... more Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the in...
Transfusion, 2012
Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology... more 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. 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). 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). 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.
Transfusion, 2011
BACKGROUND-Safe, accurate methods permitting simultaneous and/or repeated measurement of red bloo... more 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).
Transfusion, 2010
BACKGROUND-To investigate the pathophysiology of anemia and responses to RBC transfusions and ery... more BACKGROUND-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.
Transfusion, May 16, 2018
The current reference method in the U.S.A. for measuring in vivo population red blood cell (RBC) ... more The current reference method in the U.S.A. for measuring in vivo population red blood cell (RBC) kinetics utilizes 51-chromium (51 Cr) RBC labeling for determining red cell volume (RCV), 24hour post-transfusion RBC recovery (PTR 24), and long-term red cell survival (RCS). Here we provide evidence supporting adoption of a method for kinetics that uses the biotin labeled RBC *
Transfusion, Jan 16, 2018
The current reference method in the United States for measuring in vivo population red blood cell... more The current reference method in the United States for measuring in vivo population red blood cell (RBC) kinetics utilizes chromium-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 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 pharm...
Antimicrobial agents and chemotherapy, Jan 25, 2018
The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a s... more The highly variable pharmacokinetics of β-lactam antibiotics and β-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of their plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous determination of multiple β-lactam antibiotics and β-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin and tazobactam in human plasma was developed and rigorously validated according to the FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using a step-wise gradient elution. Analytes were monitored using MS/MS with a turbo ion spray source in positive ...
Transfusion, Jan 29, 2017
Critically ill preterm very-low-birthweight (VLBW) neonates (birthweight ≤ 1.5 kg) frequently dev... more 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 (PTR24 ) 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 PTR24 with storage. We hypothesized that PTR24 of allogeneic RBCs transfused to anemic VLBW newborns: 1) will be greater than PTR24 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. PTR24 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 PTR24 versus duration of RBC storage. For VLBW newborns, the ...
Transfusion, Jun 5, 2017
Biotin-labeled red blood cells (BioRBCs) are used for in vivo kinetic studies. Because BioRBC dos... more 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. 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. 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...
Transfusion, 2014
The quality of transfused red blood cells (RBCs) to treat anemia depends on its potential for oxy... more 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. 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. 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. MRL overcomes limitations of previously proposed TRCS parameters, is simpler to calculate, and is physiologically and clinically more appropriate.
AJP: Regulatory, Integrative and Comparative Physiology, 2013
A population pharmacokinetics/pharmacodynamic (PK/PD) model was developed to describe changes in ... more A population pharmacokinetics/pharmacodynamic (PK/PD) model was developed to describe changes in erythropoiesis as a function of plasma erythropoietin (EPO) concentration over the first 30 days of life in preterm infants who developed severe anemia requiring red blood cell (RBC) transfusion. Several covariates were tested as possible factors influencing the responsiveness to EPO. Discarded blood samples in 27 ventilated preterm infants born at 24-29 wk of gestation were used to construct plasma EPO, hemoglobin (Hb), and RBC concentration-time profiles. The amount of Hb removed for laboratory testing and that transfused throughout the study period were recorded. A population PK/PD model accounting for the dynamic Hb changes experienced by these infants was simultaneously fitted to plasma EPO, Hb, and RBC concentrations. A covariate analysis suggested that the erythropoietic efficacy of EPO is increased for preterm infants at later gestational ages. The PD analysis showed a sevenfold difference in maximum Hb production rate dependent on gestational age and indicated that preterm infants, when stimulated by EPO, have the capacity to produce additional Hb that may result in a decrease in RBC transfusions. The present model has utility in clinical trial simulations investigating the treatment potential of erythropoietic stimulating agents in the treatment of anemia of prematurity.
Pediatric Research, 2017
Prior conclusions that autologous neonatal red blood cells (RBC) have substantially shorter lifes... more Prior conclusions that autologous neonatal red blood cells (RBC) have substantially shorter lifespans than allogeneic adult RBCs were not based on direct comparison of autologous neonatal vs. allogeneic adult RBCs performed concurrently in the same infant. Biotin labeling of autologous neonatal RBCs and allogeneic adult donor RBCs permits concurrent direct comparison of autologous vs. allogeneic RBC lifespan. RBCs from 15 allogeneic adult donors and from 15 very low birth weight (VLBW) neonates were labeled at separate biotin densities and transfused simultaneously into the 15 neonates. Two mathematical models that account for the RBC differences were employed to estimate lifespans for the two RBC populations. Mean±SD lifespan for adult allogeneic RBC was 70.1±19.1 d, which is substantially shorter than the 120 d lifespan of both autologous and adult allogeneic RBC in healthy adults. Mean±SD lifespan for neonatal RBC was 54.2±11.3 d, which is only about 30% shorter than that of the adult allogeneic RBCs. This study provides evidence that extrinsic environmental factors primarily determine RBC survival (e.g., small bore of the capillaries of neonates, rate of oxygenation/deoxygenation cycles) rather than factors intrinsic to RBC.Pediatric Research (2017); doi:10.1038/pr.2017.14.
Endocrinology, 1998
Leydig cells are located in the interstitium of the testis and function as the primary site for t... more Leydig cells are located in the interstitium of the testis and function as the primary site for testosterone biosynthesis. Leydig cell development and steroidogenic function are dependent upon pituitary-derived LH. Circulating LH levels in prepubertal mammals are low but rise sharply during puberty, inducing terminal differentiation of immature Leydig cells into adult Leydig cells. The molecular mechanisms involved in LH action on differentiation specific gene expression and initiation of steroidogenic function in immature Leydig cells are poorly understood. Members of the CCAAT/enhancer-binding protein (C/EBP) family of basic region/leucine zipper transcription factors have previously been implicated as regulators of terminal differentiation in several cell types. In the present study we have investigated the possible involvement of C/EBP proteins in regulating LH-dependent gene expression in Leydig cells. We have detected the expression of one family member, C/EBPbeta, in Leydig cells. C/EBPbeta messenger RNA and protein levels were significantly higher in mature adult Leydig cells than in immature cells, displaying an expression pattern similar to those of other developmentally regulated genes in Leydig cells such as steroidogenesis acute regulatory (StAR) protein and 3beta-hydroxysteroid dehydrogenase. C/EBPbeta messenger RNA and protein levels also increased when immature Leydig cells were treated with either hCG, a functional analog of LH (hCG/LH), or (Bu)2cAMP. To confirm that hCG/LH and (Bu)2cAMP were acting specifically on Leydig cells, we studied their effects on C/EBPbeta expression in an established Leydig cell line (MA-10). hCG and (Bu)2cAMP treatment also induced the expression of C/EBPbeta and StAR in MA-10 cells, coincident with stimulation of steroid production in these cells. (Bu)2cAMP treatment did not alter the subcellular localization of C/EBPbeta protein in MA-10 cells, suggesting that the increase is due to stimulation of C/EBPbeta expression. We conclude that expression of C/EBPbeta is regulated by hCG/LH in Leydig cells and that C/EBPbeta may play a significant role in LH-regulated Leydig cell differentiation and function.
The AAPS journal, Jan 26, 2015
Postnatal hemoglobin (Hb) production in anemic preterm infants is determined by several factors i... more Postnatal hemoglobin (Hb) production in anemic preterm infants is determined by several factors including the endogenous erythropoietin levels, allogeneic RBC transfusions administered to treat anemia, and developmental age. As a result, their postnatal Hb production rate can vary considerably. This work introduces a novel Hb mass balance-based semiparametric approach that utilizes infant blood concentrations of Hb from the first 30 postnatal days to estimate the amount of Hb produced and the erythropoiesis rate in newborn infants. The proposed method has the advantage of not relying on specific structural pharmacodynamic model assumptions to describe the Hb production, but instead utilizes simple mass balance principles and nonparametric regression analysis. The developed method was applied to the Hb data from 79 critically ill anemic very low birth weight preterm infants to evaluate the dynamic changes in erythropoiesis during the first month of life and to determine the inter-sub...
The Journal of pediatrics, Jan 9, 2015
Based on the hypothesis that neonatal autologous red blood cell (RBC) survival (RCS) is substanti... more Based on the hypothesis that neonatal autologous red blood cell (RBC) survival (RCS) is substantially shorter than adult RBC, we concurrently tracked the survival of transfused biotin-labeled autologous neonatal and allogeneic adult RBC into ventilated, very low birth weight infants. RBC aliquots from the first clinically ordered, allogeneic adult RBC transfusion and from autologous infant blood were labeled at separate biotin densities (biotin-labeled RBC [BioRBC]) and transfused. Survival of these BioRBCs populations were concurrently followed over weeks by flow cytometric enumeration using leftover blood. Relative tracking of infant autologous and adult allogeneic BioRBC was analyzed by linear mixed modeling of batched weekly data. When possible, Kidd antigen (Jka and Jkb) mismatches between infant and donor RBCs were also used to track these 2 populations. Contrary to our hypothesis, concurrent tracking curves of RCS of neonatal and adult BioRBC in 15 study infants did not diffe...
The AAPS journal, Jan 28, 2015
Fetal RBCs are produced during a period of very rapid growth and stimulated erythropoiesis under ... more 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 erythro...
Journal of Clinical Laboratory Analysis, 2015
Reticulocyte hemoglobin content (RET-He)-an established indicator of iron status in children and ... more Reticulocyte hemoglobin content (RET-He)-an established indicator of iron status in children and adults-was determined in very low birth weight (VLBW) infants. Longitudinal retrospective RET-He data in 26 VLBW neonates during the first month of age were compared with: (a) concurrent complete blood counts (CBCs), including hemoglobin (Hb) concentration, reticulocyte count, and immature reticulocyte fraction (IRF), and erythropoietin (EPO) levels; (b) clinical variables; and (c) RET-He data from the literature for term infants, children, and adults. RET-He within 24 hr following birth was 31.8 ± 1.1 pg (mean ± SEM). This was followed by an abrupt, significant decline to 28.3 ± 1.1 pg at 2-4 days, and to steady state levels of 28.4 ± 0.5 pg thereafter. The changes in RET-He were mirrored by changes in plasma EPO, reticulocyte count, and IRF, but not Hb. Steady state RET-He values after 4 days were significantly lower than RET-He values for term infants, children, and adults (31.6 ± 0.11, 32.0 ± 0.12, and 33.0 ± 0.13 pg, respectively). Although RET-He values in VLBW infant were lower than term infants, children, and adults, the significance and mechanism(s) responsible are unknown. The present VLBW infant data are relevant to investigations assessing hemoglobinization following treatment with recombinant human EPO (r-HuEPO) and/or iron.
Pediatric Research, 2013
Anemia, a common condition among critically ill premature infants, is affected by red blood cell ... more 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). 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. 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. 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.
BMC genomics, Jan 27, 2005
Hematopoiesis is a complex developmental process controlled by a large number of factors that reg... more Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the in...
Transfusion, 2012
Measurement of red blood cell (RBC) survival (RCS) is important for investigating pathophysiology... more 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. 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). 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). 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.
Transfusion, 2011
BACKGROUND-Safe, accurate methods permitting simultaneous and/or repeated measurement of red bloo... more 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).
Transfusion, 2010
BACKGROUND-To investigate the pathophysiology of anemia and responses to RBC transfusions and ery... more BACKGROUND-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.