Metabolic impact of red blood cell exchange with rejuvenated red blood cells in sickle cell patients.

BACKGROUND: Red blood cell exchange (RCE) transfusions are a mainstay in the treatment of sickle cell anemia (SCA), and allow a temporary restoration of physiological parameters with respect to erythrocyte oxygen carrying capacity and systems metabolism. Recently, we noted that 1) RCE significantly impacts recipients' metabolism in SCA; 2) fresh and end-of-storage red blood cell (RBC) units differently impact systems of metabolism in healthy autologous recipients; and 3) phosphate/inosine/pyruvate/adenine (PIPA) solution reverses the metabolic age of stored RBCs. Therefore, we hypothesized that RCE with PIPA-treated RBC units could further increase the metabolic benefits of RCE in SCA patients. STUDY DESIGN AND METHODS: Circulating plasma and erythrocytes were collected from patients with SCA before and after RCE, with either conventional or PIPA-treated RBC units, prior to metabolomics analyses. RESULTS: Consistent with prior work, RCE significantly decreased circulating levels of markers of systemic hypoxemia (lactate, succinate) and decreased plasma levels of acyl-carnitines and amino acids. However, PIPA-treated exchanges were superior to untreated RCEs, with a higher energy state and an increased capacity to activate the pentose phosphate pathway and glutamine metabolism. In addition, RBCs and plasma from recipients of PIPA-treated RBC units resulted in significantly decreased levels of post-transfusion plasticizers, though at the expense of higher circulating levels of oxidized purines (hypoxanthine, xanthine, and the antioxidant urate). CONCLUSION: Transfusion of PIPA-treated RBCs further increases the metabolic benefits of RCE to patients with SCA, significantly reducing the levels of post-transfusion plasticizers.

Metabolomics evaluation of early-storage red blood cell rejuvenation at 4°C and 37°C.

BACKGROUND: Refrigerated red blood cell (RBC) storage results in the progressive accumulation of biochemical and morphological alterations collectively referred to as the storage lesion. Storage-induced metabolic alterations can be in part reversed by rejuvenation practices. However, rejuvenation requires an incubation step of RBCs for 1 hour at 37°C, limiting the practicality of providing "on-demand," rejuvenated RBCs. We tested the hypothesis that the addition of rejuvenation solution early in storage as an adjunct additive solution would prevent-in a time window consistent with the average age of units transfused to sickle cell recipients at Duke (15 days)-many of the adverse biochemical changes that can be reversed via standard rejuvenation, while obviating the incubation step. STUDY DESIGN AND METHODS: Metabolomics analyses were performed on cells and supernatants from AS-1 RBC units (n = 4), stored for 15 days. Units were split into pediatric bag aliquots and stored at 4°C. These were untreated controls, washed with or without rejuvenation, performed under either standard (37°C) or cold (4°C) conditions. RESULTS: All three treatments removed most metabolic storage by-products from RBC supernatants. However, only standard and cold rejuvenation provided significant metabolic benefits as judged by the reactivation of glycolysis and regeneration of adenosine triphosphate and 2,3-diphosphoglycerate. Improvements in energy metabolism also translated into increased capacity to restore the total glutathione pool and regenerate oxidized vitamin C in its reduced (ascorbate) form. CONCLUSION: Cold and standard rejuvenation of 15-day-old RBCs primes energy and redox metabolism of stored RBCs, while providing a logistic advantage for routine blood bank processing workflows.

Estimation of Achievable Oxygen Consumption Following Transfusion With Rejuvenated Red Blood Cells.

Erythrocyte storage induces a nonphysiological increase in hemoglobin-oxygen affinity (quantified by low p50, the oxygen tension at 50% hemoglobin saturation), which can be restored through biochemical rejuvenation. The objective was to mathematically model the impact of transfusing up to 3 standard allogeneic units or rejuvenated units on oxygen delivery (DO2) and oxygen consumption (VO2). Oxygen dissociation curves were generated from additive solution-1 red blood cell (RBC) leukoreduced units (n = 7) before and after rejuvenation following manufacturer's instructions. Two of these units were used to prepare standard or rejuvenated donor RBC and added to samples of fresh whole blood. These admixtures were used to construct an in vitro transfusion model of postoperative anemia and determine a linear equation for calculating the sample p50, which was subsequently used to calculate DO2 and VO2 after simulated transfusions. Whole blood-packed red blood cell unit admixture p50s could be predicted from a linear model including the p50 of its components, the mass fraction of the transfused component, and interaction terms (R2 = .99, P < 0.001). Transfusion with standard units slightly, but significantly, increased projected DO2 compared with rejuvenated units (P = 0.03), but rejuvenated units markedly increased projected VO2 (P = 0.03). Standard units did not significantly change VO2 relative to pre-transfusion levels (P > 0.1). Using high-p50, rejuvenated RBC in simulated transfusions greatly improved projected VO2, indicating the potential for increased end-organ oxygen availability compared with standard transfusion. Patient capacity to increase cardiac output after cardiac surgery may be limited. Transfusing high-p50 RBC in this setting may improve the perioperative care of these patients.

Performance of a gravitational marrow separator, multidirectional bone marrow aspiration needle, and repeated bone marrow collections on the production of concentrated bone marrow and separation of mesenchymal stem cells in horses.

Objective-To determine the efficiency of a novel point-of-care gravitational marrow separator and bone marrow aspiration needle for concentrated bone marrow production and bone marrow-derived mesenchymal stem cell (MSC) separation and assess the effect of repeated bone marrow collections in horses. Animals-8 healthy adult horses. Procedures-Bone marrow aspiration was performed twice (1 month apart) from sternebral bodies with a standard or prototype multidirectional needle. Concentrated bone marrow was obtained by gravitational marrow separation and evaluated for WBC and platelet counts, automated and cytomorphologic cell differential counts, MSCs, and cell viability. Results-Concentrated bone marrow samples obtained with the marrow separator had 5- to 19-fold bone marrow-derived MSC, WBC, and platelet counts, compared with original bone marrow samples. Use of a multidirectional needle increased the frequency of obtaining MSC-richer concentrated bone marrow. Repeating bone marrow aspiration at 1 month yielded greater MSC numbers but slightly lower cell viability after processing. Conclusions and Clinical Relevance-The gravitational bone marrow separator and multidirectional needle were used to effectively harvest bone marrow and improve the quality of concentrated bone marrow. Comparable, or even greater, numbers of bone marrow-derived MSCs were collected by repeated bone marrow aspiration after a 1-month interval from the same aspiration sites. Use of the marrow separator and multidirectional bone marrow aspiration needle can facilitate a 1-step, point-of-care, nonlaboratory method to obtain concentrated bone marrow as a mixture of bone marrow-derived MSCs and growth factors from platelets and plasma.

Contrast-enhanced micro-computed tomography of fatigue microdamage accumulation in human cortical bone.

Conventional methods used to image and quantify microdamage accumulation in bone are limited to histological sections, which are inherently invasive, destructive, two-dimensional, and tedious. These limitations inhibit investigation of microdamage accumulation with respect to volumetric spatial variation in mechanical loading, bone mineral density, and microarchitecture. Therefore, the objective of this study was to investigate non-destructive, three-dimensional (3-D) detection of microdamage accumulation in human cortical bone using contrast-enhanced micro-computed tomography (micro-CT), and to validate micro-CT measurements against conventional histological methods. Unloaded controls and specimens loaded in cyclic uniaxial tension to a 5% and 10% reduction in secant modulus were labeled with a precipitated BaSO4 stain for micro-CT and basic fuchsin for histomorphometry. Linear microcracks were similarly labeled by BaSO4 and basic fuchsin as shown by backscattered electron microscopy and light microscopy, respectively. The higher X-ray attenuation of BaSO4 relative to the bone extracellular matrix provided enhanced contrast for the detection of damage that was otherwise not able to be detected by micro-CT prior to staining. Therefore, contrast-enhanced micro-CT was able to nondestructively detect the presence, 3-D spatial location, and accumulation of fatigue microdamage in human cortical bone specimens in vitro. Microdamage accumulation was quantified on segmented micro-CT reconstructions as the ratio of BaSO4 stain volume (SV) to total bone volume (BV). The amount of microdamage measured by both micro-CT (SV/BV) and histomorphometry (Cr.N, Cr.Dn, Cr.S.Dn) progressively increased from unloaded controls to specimens loaded to a 5% and 10% reduction in secant modulus (p < 0.001). Group means for micro-CT measurements of damage accumulation were strongly correlated to those using histomorphometry (p < 0.05), validating the new methods. Limitations of the new methods in the present study included that the precipitated BaSO4 stain was non-specific and non-biocompatible, and that micro-CT measurements exhibited greater variability compared to conventional histology. Nonetheless, contrast-enhanced micro-CT enabled non-destructive imaging and 3-D spatial information, which are not possible using conventional histological methods.

Detection of dentinal cracks using contrast-enhanced micro-computed tomography.

A new technique using contrast enhanced micro-computed tomography (micro-CT) was developed to improve the ability to detect dentinal cracks in teeth and assess associated risks to oral health. Extracted, whole human molars that exhibited visual evidence of external cracks following extraction and machined, partially fractured elephant dentin specimens were labeled by BaSO(4) precipitation and imaged by micro-CT. Contrast-enhanced micro-CT was demonstrated in vitro to enable non-destructive, 3-D imaging of the presence, morphology and spatial location of dentinal cracks in whole human molars and machined specimens. BaSO(4) staining provided enhanced contrast for the detection of cracks that could not be detected prior to staining. Backscattered SEM micrographs showed that BaSO(4) was precipitated on the surfaces of dentinal cracks and within adjacent tubules. The new methods demonstrated in this study are expected to be useful for clinical and scientific studies investigating the etiology and treatment of dentinal cracks in teeth.

Anatomic variation in the elastic anisotropy of cortical bone tissue in the human femur.

Experimental investigations for anatomic variation in the magnitude and anisotropy of elastic constants in human femoral cortical bone tissue have typically focused on a limited number of convenient sites near the mid-diaphysis. However, the proximal and distal ends of the diaphysis are more clinically relevant to common orthopaedic procedures and interesting mechanobiology. Therefore, the objective of this study was to measure anatomic variation in the elastic anisotropy and inhomogeneity of human cortical bone tissue along the entire length (15%-85% of the total femur length), and around the periphery (anterior, medial, posterior and lateral quadrants) of the femoral diaphysis, using ultrasonic wave propagation in the three orthogonal specimen axes. The elastic symmetry of tissue in the distal and extreme proximal portions of the diaphysis (15%-45% and 75%-85% of the total femur length, respectively) was, at most, orthotropic. In contrast, the elastic symmetry of tissue near the mid- and proximal mid-diaphysis (50%-70% of the total femur length) was reasonably approximated as transversely isotropic. The magnitudes of elastic constants generally reached maxima near the mid- and proximal mid-diaphysis in the lateral and medial quadrants, and decreased toward the epiphyses, as well as the posterior and anterior quadrants. The elastic anisotropy ratio in the longitudinal and radial anatomic axes showed the opposite trends. These variations were significantly correlated with the apparent tissue density, as expected. In summary, the human femur exhibited statistically significant anatomic variation in elastic anisotropy, which may have important implications for whole bone numerical models and mechanobiology.

Systematic error in mechanical measures of damage during four-point bending fatigue of cortical bone.

Accumulation of fatigue microdamage in cortical bone specimens is commonly measured by a modulus or stiffness degradation after normalizing tissue heterogeneity by the initial modulus or stiffness of each specimen measured during a preloading step. In the first experiment, the initial specimen modulus defined using linear elastic beam theory (LEBT) was shown to be nonlinearly dependent on the preload level, which subsequently caused systematic error in the amount and rate of damage accumulation measured by the LEBT modulus degradation. Therefore, the secant modulus is recommended for measurements of the initial specimen modulus during preloading. In the second experiment, different measures of mechanical degradation were directly compared and shown to result in widely varying estimates of damage accumulation during fatigue. After loading to 400,000 cycles, the normalized LEBT modulus decreased by 26% and the creep strain ratio decreased by 58%, but the normalized secant modulus experienced no degradation and histology revealed no significant differences in microcrack density. The LEBT modulus was shown to include the combined effect of both elastic (recovered) and creep (accumulated) strain. Therefore, at minimum, both the secant modulus and creep should be measured throughout a test to most accurately indicate damage accumulation and account for different damage mechanisms. Histology revealed indentation of tissue adjacent to roller supports, with significant sub-surface damage beneath large indentations, accounting for 22% of the creep strain on average. The indentation of roller supports resulted in inflated measures of the LEBT modulus degradation and creep. The results of this study suggest that investigations of fatigue microdamage in cortical bone should avoid the use of four-point bending unless no other option is possible.