Herrick, James L.

Abstract Background Hemoglobin S (HbS) causes sickling disorders. In the laboratory, confirmation of HbS in patient samples with varying percentages is essential. Assessment is routinely performed using capillary electrophoresis (CE) and/or high-performance liquid chromatography. Sickle solubility (SS) exploits decreased solubility of HbS under reducing conditions and is a confirmatory test. SS is unreliable with low HbS, elevated HbF, severe anemia, erythrocytosis, hyperglobulinemia, extreme leukocytosis, hyperlipidemia, or increased serum proteins. Infant samples commonly have low percentages of HbS, high HbF, and limited sample volume. Intact globin chain Mass Spectrometry (MS) is used to identify abnormal hemoglobin variants by detecting the mass change (normal α/β/γ mass =15127/15867/15996 amu), respectively). MS has potential advantages in sensitivity and efficiency over SS and requires lower input sample volume. Herein we compare the utility of MS versus SS for confirmation of HbS. Methods Twenty-three EDTA-anticoagulated blood samples containing low percentages of HbS were identified. SS was performed on 30μL washed packed cells (requires 200μL whole blood) per standard methods. Intact Quadrupole Time-of-Flight MS (Accurate-Mass Q-TOF LC/MS 6520, Agilent Technologies, California) was performed using 10μL whole blood sample in acetonitrile solution reagent and results were compared. HbS and HbF levels were assayed by capillary electrophoresis (Sebia, France) per standard methods. Results Select results are summarized in Table 1. SS detected HbS percentages to 4.8% when HbF was low but not 6% HbS when Hb F is high. MS showed no HbF interference and identified HbS peaks (at 15837 amu) down to 3.1%. HbS percentages <3.1 were undetected by both methods. Conclusions Overall, MS was superior to SS for confirming HbS, particularly in samples with low variant percentage and/or elevated HbF. MS is also optimal when sample volume is minimal, such as for infants. Percentages of HbS <3% require methods such as IEF or DNA sequencing for confirmation.

AbstractForensic fractographic features of bone reliably establish crack propagation in perimortem injuries. We investigated if similar fracture surface features characterize postmortem fractures. Experimentally induced peri‐ and postmortem fractures were used to assess if fractographic features vary as bone elasticity decreases during the postmortem interval (PMI). Thirty‐seven unembalmed, defleshed human femoral shafts from males and females aged 33–81 years were fractured at varying PMIs with a drop test frame using a three‐point bending setup and recorded with a high‐speed camera. Vital statistics, cause of death, PMI length, temperature, humidity, collagen percentage, water loss, fracture energy, and fractography scores were recorded for each sample. Results showed that fractographic features associated with perimortem fractures were expressed in PMIs up to 40,600 accumulated degree hours (ADH), or 60 warm weather days. Hackle was the most consistently expressed feature, occurring in all fractures regardless of ADH. The most variable characteristics were wake features (78.4%) and arrest ridges (70.3%). Collagen percentage did not correlate strongly with ADH (r = −0.04, p = 0.81); however, there was a strong significant correlation between ADH and water loss (r = 0.74, p < 0.001). Multinomial logistic regression showed no association between fractographic feature expression and ADH or collagen percentage. In conclusion, forensic fractographic features reliably determine initiation and directionality of crack propagation in experimentally induced PMIs up to 40,600 ADH, demonstrating the utility of this method into the recent postmortem interval. This expression of reliable fractographic features throughout the early PMI intimates these characteristics may not be useful standalone features for discerning peri‐ versus postmortem fractures.

10531 Background: Osteosarcoma (OS) is a clinically challenging primary malignant bone tumor that has a high rateof local recurrence and metastasis. Disease free survival has been reported to be as low as 19% despite contemporary chemotherapy regimens and surgery. Wnt signaling is known to play a role in osteogenesis and cancer pathogenesis, therefore is a promising target for therapeutic intervention in OS. Secreted Frizzled Related Proteins (sFRPs), Dickkopf-1 (DKK1), and Sclerostin (SOST) are secreted Wnt inhibitor proteins that regulate Wnt signaling in bone. This study investigates the activity of sFRP3, DKK1, and SOST in serum from OS patients with the goal of developing therapeutic Wnt inhibitors and clinically useful biomarkers. Methods: Enzyme linked immunosorbent assayswere used to quantify sFRP3, DKK1 and SOST levels in human serum (n=40 pairs; 20 OS patients, 20 aged matched non-diseased controls). Patients were stratified into 2 age groups: >35 (n=26 pairs) and <30 (n=14 pairs) years of age. Clinical data from the medical records was correlated with experimental results. Using a Wilcoxon signed rank test, a p–value <0.05 was significant. Results: sFRP3 levels were significantly decreased in 55% (22/40) of diseased samples compared to 20% (8/40) of the non-diseased controls (p= 0.007). DKK1 levels were elevated in 40% (16/40) of OS samples compared to 30% (12/40) of the non-diseased controls (p= 0.08). SOST levels were increased in 42.5% (17/40) of diseased samples compared to 32.5% (13/40) of the non-diseased controls (p= 0.38). When stratified by age, the younger patients showed a statistically significant decrease in sFRP3 expression (p<0.001). Serum levels of sFRP3, DKK1, and SOST were not statistically significant between metastatic and non-metastatic OS. Conclusions: Serum sFRP3 levels were significantly decreased in OS patients, a difference that was even more pronounced in the pediatric strata. Since sFRP3 is differentially expressed in patient serum it has potential as a clinical biomarker for initial tumor diagnosis and early detection of recurrence. Decreased sFRP3 expression may be responsible for the up regulation of Wnt signaling in OS. Wnt inhibitors such as sFRP3 have potential as therapeutic agents in OS warranting further investigation.

AbstractTo investigate the effect of sustained bone morphogenetic protein‐2 (BMP‐2) release kinetics on bone formation in poly(propylene fumarate) (PPF) scaffolds, different poly(lactic‐co‐glycolic acid) (PLGA) microspheres were used as delivery vehicles. All PPF scaffolds had the same 75% porous structure, while the degradation rate of the embedded PLGA microspheres was changed to tailor BMP‐2 release by varying the lactic‐to‐glycolic acid (L:G) ratio in the copolymer. Four PLGA microsphere formulations with 50/50, 65/35, 75/25, and 85/15 L:G ratios and varying in vivo degradation rates were fabricated. The in vitro and in vivo BMP‐2 release kinetics were determined by analyzing radiolabeled 125I‐BMP‐2. Biological activity of released BMP‐2 was tested using a W20‐17 cell culture model in vitro and a subcutaneous rat model in vivo. Corresponding outcome parameters were defined as capacity to increase the in vitro AP activity in weekly consecutive cell cultures over 14 weeks and the in vivo bone formation after 7 and 14 weeks. The PLGA/PPF composites showed similar biological activity and BMP‐2 release profiles in vitro. In vivo, PPF/PLGA 85:15 composite released significantly less BMP‐2 per time point in the first weeks. Micro‐CT and histological analysis after 7 and 14 weeks of implantation showed bone formation, which significantly increased over time for all composites. No significant differences were seen between the composites. Overall, the results of this study show that small differences in BMP‐2 sustained release had no significant effect on BMP‐2 osteogenic efficacy in PPF/PLGA composites. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 477–487, 2018.