Reticulocyte hemoglobin equivalent as a marker to assess iron deficiency: A large pediatric tertiary care hospital study.

INTRODUCTION: Detection of iron deficiency (ID) remains challenging. We aimed to evaluate the performance of reticulocyte hemoglobin equivalent (Ret-He) as a potential diagnostic marker to assess ID and iron deficiency anemia (IDA) in a large pediatric cohort. METHODS: A total of 3158 patients (aged 15 days to 19 years with a median age of 8.5 years; 60.2% female) were retrospectively studied. Statistical analysis was performed (a) to evaluate relationship of Ret-He with other relevant complete blood count and iron panel parameters; (b) to compare the levels of Ret-He in ID and IDA groups to a control group; and (c) to assess sensitivity and specificity of Ret-He in ID, IDA, and anemia without ID groups. RESULTS: Ret-He values were significantly positively correlated to ferritin and transferrin saturation (TSAT). The median Ret-He was significantly lower in ID. A Ret-He cutoff of ≤30.0 pg distinguished cases of ID from the control group with a sensitivity of 90.2%, specificity of 59.5%, and area under curve (AUC) of 0.88. Ret-He showed better diagnostic performance in the IDA group and acceptable performance for ID without anemia. The sensitivity, specificity, and AUC were 90.1%, 80.9%, and 0.93 for IDA at cutoff value of ≤27.4 pg, and 80.8%, 51.1%, and 0.70 for ID without anemia at cutoff value of ≤30.8 pg, respectively. CONCLUSION: Our large pediatric tertiary care hospital study demonstrates that Ret-He is a reliable marker to help confirm IDA in pediatric population. However, further studies are needed for its use to capture the early stages of ID.

Unique Hemoglobinopathy Pattern Following Treatment with Voxelotor.

OBJECTIVE: Voxelotor, a FDA-approved drug for the treatment of patients with sickle cell disease (SCD), inhibits hemoglobin S (HbS) polymerization and increases total hemoglobin via hemolysis reduction. This drug has shown unique patterns in hemoglobin fractionation, affecting its interpretation. We aimed to evaluate whether these voxelotor-induced changes can be linked to improvement of hemolysis markers in pediatric patients on voxelotor. METHODS: A total of 15 patients (age 12 to 20 years; 40% females) on voxelotor were evaluated to compare changes in the hemoglobin fractionation by capillary electrophoresis, total hemoglobin, reticulocyte percentage (retic%), lactate dehydrogenase (LDH), and bilirubin measurements before and after the recorded date of voxelotor prescription. RESULTS: Hemoglobin fractionation showed changes in the profile of 60% (9/15) of the patients studied. Out of the 9 patients for which voxelotor showed changes in the hemoglobin fractionation, 44% (4/9) had an increase of >1 g/dL in their total hemoglobin after voxelotor treatment was started. Assessment of other hemolysis markers available showed decreased LDH (4/4), retic % (6/8), and bilirubin (3/4). CONCLUSIONS: Unique pattern of hemoglobin fractionation analysis following therapy with voxelotor has potential as a tool for the assessment of response and/or compliance to voxelotor for the treatment of SCD.

Pediatric vitamin D: Pseudo-hypervitaminosis.

BACKGROUND: Vitamin D toxicity is rare in pediatric population. Falsely elevated levels of 25-hydroxyvitamin D have been reported as a major challenge with immunoassay methods for quantifying vitamin D metabolites. CASE PRESENTATION AND METHOD: Here, we present two pediatric cases of falsely elevated 25-hydroxyvitamin D that resulted in unnecessary further testing. We also report significant same-day variation in the measurement of 25-hydroxyvitamin D using the Abbott i2000SR immunoassay. Samples were spun twice and their values were confirmed with the gold standard liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for confirmation. CONCLUSION: The addition of a centrifugation step prior to sample testing resolved the variation observed in the measurement of 25-hydroxyvitamin D levels. The patient samples were confirmed with instruments from a different vendor and LC-MS/MS. Re-centrifugation of samples resolved the variation in the 25-hydroxyvitamin D values.

Validation of atovaquone plasma levels by liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring in pediatric patients.

Background: Atovaquone has traditionally been used as an antiparasitic and antifungal agent, but recent studies have shown its potential as an anticancer agent. The high variability in atovaquone bioavailability highlights the need for therapeutic drug monitoring, especially in pediatric patients. The goal of our study was to develop and validate the performance of an assay to quantify atovaquone plasma concentrations collected from pediatric cancer patients using LC-MS/MS. Methods: Atovaquone was extracted from a 10 µL volume of K2-EDTA human plasma using a solution consisting of ACN: EtOH: DMF (8:1:1 v:v:v), separated using reverse-phase chromatography, and detected using a SCIEX 5500 QTrap MS system. LC-MS/MS assay performance was evaluated for precision, accuracy, carryover, sensitivity, specificity, linearity, and interferences. Results: Atovaquone and its deuterated internal standard were analyzed using a gradient chromatographic method that had an overall cycle-time of 7.4 min per injection, and retention times of 4.3 min. Atovaquone was measured over a dynamic concentration range of 0.63 - 80 µM with a deviation within ≤ ± 5.1 % of the target value. Intra- and inter-assay precision were ≤ 2.7 % and ≤ 8.4 %, respectively. Dilutional, carryover, and interference studies were also within acceptable limits. Conclusions: Our studies have shown that our LC-MS/MS-based method is both reliable and robust for the quantification of plasma atovaquone concentrations and can be used to determine the effective dose of atovaquone for pediatric patients treated for AML.

Comparison of Cystatin C and Creatinine-Based Equations with Measured Glomerular Filtration Rate in a Diverse Pediatric Population.

BACKGROUND: Accurate assessment of kidney function is essential for early detection of kidney damage. While measured glomerular filtration rate (mGFR) is occasionally used as a reference, estimated GFR (eGFR) from serum creatinine- and cystatin C (CysC)-based equations are routinely used in clinical practice as a reliable and less invasive approach. In pediatric populations, CysC-based equations provide a closer approximation as they are independent of body composition. Limited information is available on the performance of CysC-based equations in comparison with mGFR with tracers other than iohexol. Therefore, the goal of our study was to evaluate how eGFR, based on several CysC- and creatinine-based equations, with and without race correction, relates to mGFR in a diverse pediatric population. METHODS: A total of 43 patients (7 months to 21 years) from diverse race/ethnicity were retrospectively studied to compare the mGFR from multiple blood sample collections after intravenous tracer injection (Tc-99mDTPA) with eGFR using 9 equations. Deming regression analyses were performed to assess correlation between the mGFR and eGFRs. RESULTS: The average mGFR for this cohort was 95.0 mL/min/1.73 m2. Race-corrected (RC) equations gave overestimated eGFR across all ethnic groups, with the lowest bias for Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) CysC-creatinine (34.14 mL/min/1.73 m2). The best correlations to mGFR, percentage of eGFR within 30% of mGFR (P30), and lowest biases were from non-race-corrected (NRC) equations Chronic Kidney Disease in Children (CKiD) (0.6460, 65.1%, 2.86 mL/min/1.73 m2), CKD-EPI CysC (0.6858, 69.8%, 11.01 mL/min/1.73 m2), and Schwartz CysC (0.6876, 79.1%, -14.00 mL/min/1.73 m2). CONCLUSION: Overall, CysC-based equations without race correction provide a good approximation of mGFR and a less invasive alternative to monitoring kidney function in pediatric population, irrespective of race/ethnicity.

True hyponatremia in the setting of pegasparagase-induced hypertriglyceridemia: A case report.

Pegasparagase (PEG-Asparaginase) induced hypertriglyceridemia is rare in the treatment of lymphoblastic lymphoma in children. We present a case of PEG-asparaginase induced hypertriglyceridemia that was incidentally identified and suspicion of its interference with sodium measurement in the clinical laboratory. The use of a direct ion selective method clarified the presence of true hyponatremia. The patient was treated with an oral Fenofibrate therapy which resolved the hypertriglyceridemia. This case highlights that PEG-asparaginanse and Olazanpine can induce hypertriglyceridemia in acute lymphoblastic leukemia and it may be useful to obtain baseline triglyceride measurements for these patients.

SARSCoV-2 serostatus amongst vaccinated employees at a pediatric hospital system.

To gain insights on the heterogeneity of immune responses to vaccination against SARS-CoV-2 and to identify factors that could make individuals vulnerable to infection due to lack of response to vaccination, our hospital started offering free voluntary post-antibody testing against the spike protein IgG for all fully vaccinated employees. Post-vaccination response against SARS-CoV-2 was assessed using the FDA-EUA approved VITROS anti-SARS-CoV-2 IgG immunometric assay specific to the spike protein. Out of a total of 3266 antibody tests performed in fully vaccinated Texas Children's, 99.4% had a positive antibody response to the spike protein. From the 21 employees (0.6%) that had a negative response, 66.7% reported taking immunosuppressive drugs and/or biologics. Our data shows that most of the employees tested at our institution mounted an immune response to the immunogen in the vaccine. Post-vaccination antibody testing against SARS-CoV-2 can provide useful information to guide decisions about future vaccine doses.

Interference of eltrombopag with bilirubin measurements on the Vitros 5600 analyzer.

BACKGROUND: Eltrombopag is a thrombopoietin-receptor agonist used to restore platelet count to hemostatic levels in chronic immune thrombocytopenia. The drug has shown to have hepatobiliary adverse effects, but also positive interference with the analytical measurement of bilirubin. Understanding the degree of interference of this drug with bilirubin testing becomes relevant in the clinical management of these patients. METHODS: Eltrombopag at concentrations ranging from 10 to 150 µg/ml was spiked into plasma samples with different baseline concentrations of bilirubin. Total bilirubin, conjugated, and unconjugated bilirubin were measured for each sample using VITROS TBILI and BuBc slides on the Vitros 5600 automated chemistry platform, and interference was assessed. RESULTS: Plasma samples spiked with eltrombopag yielded falsely elevated bilirubin measurements compared to baseline, with the degree of elevation increasing with greater concentrations of eltrombopag. Bilirubin values were increased relative to baseline across all groups, except in conjugated bilirubin measurements in samples with low baseline conjugated bilirubin. For samples with low total bilirubin at baseline, >100 µg/ml of eltrombopag resulted in an error of >+0.6 mg/dl on the measured total bilirubin. For samples with low unconjugated bilirubin at baseline, the error for the same concentrations was >+0.7 mg/dl. CONCLUSION: Our results show that, at supra-physiologically high concentrations, eltrombopag can positively interfere with bilirubin measurements on Vitros 5600 platform.

Hemoglobin fractionation by Sebia Capillarys 2 Flex Piercing System as primary method for evaluation of hemoglobinopathies.

BACKGROUND: Hemoglobin fractionation by capillary zone electrophoresis (CE) is becoming a popular method for the identification of hemoglobin variants that can cause hemoglobinopathies. The goal of this study was to compare the performance of capillary electrophoresis using Sebia Capillarys 2 Flex Piercing system (CE-S) with high-pressure liquid chromatography (HPLC) using Primus Ultra2 Resolution Variants System (HPLC-P) as a primary method in hemoglobinopathy work-up. METHODS: A total of 306 blood specimens submitted for evaluation of hemoglobinopathies were studied using HPLC-P and CE-S. RESULTS: The reference ranges for Hb A, A2 and F agreed well between methods. All common variants containing Hb S and Hb C were detected by both methods. Quantification of Hb A2 with HPLC-P required a correction in the presence of Hb S, while quantification of Hb A2 was slightly overestimated by CE-S in the presence of Hb C. Of 41 samples containing other variants, 2 were not identified by HPLC-P and 3 were not identified by CE-S. CONCLUSION: CE-S provides comparable information to that obtained by HPLC-P and it is a reliable primary method for the evaluation of hemoglobin variants.

A Human Vascular Injury-on-a-Chip Model of Hemostasis.

Hemostasis is an innate protective mechanism that plays a central role in maintaining the homeostasis of the vascular system during vascular injury. Studying this essential physiological process is often challenged by the difficulty of modeling and probing the complex dynamics of hemostatic responses in the native context of human blood vessels. To address this major challenge, this paper describes a microengineering approach for in vitro modeling of hemostasis. This microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that is actuated with spatial precision to simulate penetrating vascular injuries. The system recapitulates key features of the hemostatic response to acute vascular injury as observed in vivo, including i) thrombin-driven accumulation of platelets and fibrin, ii) formation of a platelet- and fibrin-rich hemostatic plug that halts blood loss, and iii) matrix deformation driven by platelet contraction for wound closure. Moreover, the potential use of this model for drug testing applications is demonstrated by evaluating the effects of anticoagulants and antiplatelet agents that are in current clinical use. The vascular injury-on-a-chip may serve as an enabling platform for preclinical investigation of hematological disorders and emerging therapeutic approaches against them.

Interrelationships between structure and function during the hemostatic response to injury.

Extensive studies have detailed the molecular regulation of individual components of the hemostatic system, including platelets, coagulation factors, and regulatory proteins. Questions remain, however, about how these elements are integrated at the systems level within a rapidly changing physical environment. To answer some of these questions, we developed a puncture injury model in mouse jugular veins that combines high-resolution, multimodal imaging with functional readouts in vivo. The results reveal striking spatial regulation of platelet activation and fibrin formation that could not be inferred from studies performed ex vivo. As in the microcirculation, where previous studies have been performed, gradients of platelet activation are readily apparent, as is an asymmetrical distribution of fibrin deposition and thrombin activity. Both are oriented from the outer to the inner surface of the damaged vessel wall, with a greater extent of platelet activation and fibrin accumulation on the outside than the inside. Further, we show that the importance of P2Y12 signaling in establishing a competent hemostatic plug is related to the size of the injury, thus limiting its contribution to hemostasis to specific physiologic contexts. Taken together, these studies offer insights into the organization of hemostatic plugs, provide a detailed understanding of the adverse bleeding associated with a widely prescribed class of antiplatelet agents, and highlight differences between hemostasis and thrombosis that may suggest alternative therapeutic approaches.

Bax-induced apoptosis shortens the life span of DNA repair defect Ku70-knockout mice by inducing emphysema.

Cells with DNA damage undergo apoptosis or cellular senescence if the damage cannot be repaired. Recent studies highlight that cellular senescence plays a major role in aging. However, age-associated diseases, including emphysema and neurodegenerative disorders, are caused by apoptosis of lung alveolar epithelial cells and neurons, respectively. Therefore, enhanced apoptosis also promotes aging and shortens the life span depending on the cell type. Recently, we reported that ku70(-) (/) (-)bax(-) (/) (-) and ku70(-) (/) (-)bax(+/) (-) mice showed significantly extended life span in comparison with ku70(-) (/) (-)bax(+/+) mice. Ku70 is essential for non-homologous end joining pathway for DNA double strand break repair, and Bax plays an important role in apoptosis. Our study suggests that Bax-induced apoptosis has a significant impact on shortening the life span of ku70(-) (/) (-) mice, which are defective in one of DNA repair pathways. The lung alveolar space gradually enlarges during aging, both in mouse and human, and this age-dependent change results in the decrease of respiration capacity during aging that can lead to emphysema in more severe cases. We found that emphysema occurred in ku70(-) (/) (-) mice at the age of three-months old, and that Bax deficiency was able to suppress it. These results suggest that Bax-mediated apoptosis induces emphysema in ku70(-) (/) (-) mice. We also found that the number of cells, including bronchiolar epithelial cells and type 2 alveolar epithelial cells, shows a higher DNA double strand break damage response in ku70 KO mouse lung than in wild type. Recent studies suggest that non-homologous end joining activity decreases with increased age in mouse and rat model. Together, we hypothesize that the decline of Ku70-dependent DNA repair activity in lung alveolar epithelial cells is one of the causes of age-dependent decline of lung function resulting from excess Bax-mediated apoptosis of lung alveolar epithelial cells (and their progenitor cells).

Thermally responsive nanoparticle-encapsulated curcumin and its combination with mild hyperthermia for enhanced cancer cell destruction.

In this study, thermally responsive polymeric nanoparticle-encapsulated curcumin (nCCM) was prepared and characterized. The nCCM is ≈ 22 and 300 nm in diameter at 37 and 22 °C, respectively. The smaller size of the nCCM at 37 °C was found to significantly facilitate its uptake in vitro by human prostate adenocarcinoma PC-3 cancer cells. However, the intracellular nCCM decreases rapidly (rather than plateaus) after reaching its peak at ≈ 1.5 h during a 3-day incubation of the PC-3 cells with nCCM. Moreover, a mild hyperthermia (with negligible cytotoxicity alone) at 43 °C applied between 1 and 1.5 h during the 3-day incubation not only increases the peak uptake but also alters intracellular distribution of nCCM (facilitating its delivery into cell nuclei), which helps to retain a significantly much higher level of intracellular curcumin. These effects of mild hyperthermia could be due in part to the thermal responsiveness of the nCCM: they are more positively charged at 43 °C and can be more easily attracted to the negatively charged nuclear membrane to enter nuclei as a result of electrostatic interaction. Ultimately, a combination of the thermally responsive nCCM and mild hyperthermia significantly enhances the anticancer capability of nCCM, resulting in a more than 7-fold decrease in its inhibitory concentration to reduce cell viability to 50% (IC50). Further mechanistic studies suggest injury pathways associated with heat shock proteins 27 and 70 should contribute to the enhanced cancer cell destruction by inducing cell apoptosis and necrosis. Overall, this study demonstrates the potential of combining mild hyperthermia and thermally responsive nanodrugs such as nCCM for augmented cancer therapy.

Multifunctional albumin nanoparticles as combination drug carriers for intra-tumoral chemotherapy.

Current cancer therapies are challenged by weakly soluble drugs and by drug combinations that exhibit non-uniform biodistribution and poor bioavailability. In this study, we have presented a new platform of advanced healthcare materials based on albumin nanoparticles (ANPs) engineered as tumor penetrating, delivery vehicles of combinatorially applied factors to solid tumors. These materials were designed to overcome three sequential key barriers: tissue level transport across solid tumor matrix; uptake kinetics into individual cancer cells; therapeutic resistance to single chemotherapeutic drugs. The ANPs were designed to penetrate deeper into solid tumor matrices using collagenase decoration and evaluated using a three-dimensional multicellular melanoma tumor spheroid model. Collagenase modified ANPs exhibited 1-2 orders of magnitude greater tumor penetration than unmodified ANPs into the spheroid mass after 96 hours, and showed preferential uptake into individual cancer cells for smaller sized ANPs (<100 nm). For enhanced efficacy, collagenase coated ANPs were modified with two therapeutic agents, curcumin and riluzole, with complementary mechanisms of action for combined cell cycle arrest and apoptosis in melanoma. The collagenase coated, drug loaded nanoparticles induced significantly more cell death within 3-D tumor models than the unmodified, dual drug loaded ANP particles and the kinetics of cytotoxicity was further influenced by the ANP size. Thus, multifunctional nanoparticles can be imbued with complementary size and protease activity features that allow them to penetrate solid tumors and deliver combinatorial therapeutic payload with enhanced cancer cytotoxicity but minimal collateral damage to healthy primary cells.