Female Donors Research Articles

Characterization of Pre-Existing Neutralizing Antibody to Human Adenovirus Types 5 and 49 and Simian Type 23 in Chinese Population.

Recombinant adenovirus vector has been widely used in vaccine development. Due to the pre-existing immunity of human adenovirus type 5 (HAd5) in humans, a range of rare human and chimpanzee adenovirus vectors have been developed. In the previous study, we constructed novel adenovirus vector Sad23L and Ad49L based on simian adenovirus type 23 (SAd23) and human adenovirus type 49 (HAd49), which were used in the development of ZIKV and COVID-19 vaccines. However, the levels of pre-existing neutralizing antibody (NAb) of HAd49 and SAd23 remain unclear in China. In this study, we measured NAbs titers of HAd5, HAd49, and SAd23 in 600 healthy blood donors from 6 regions across China. NAb titer of HAd49 or SAd23 was significantly lower than that of HAd5 (p < 0.001). There was no significant difference in seroprevalence and NAb titers of three adenoviruses between male and female donors. The seropositive rates of HAd5 and SAd23 increased with age growth in a positive correlation (p < 0.01), while in contrast to HAd5, HAd49, and SAd23 had a low level of pre-existing immunity in Chinese population, which suggested that Ad49L and Sad23L vectors could be used in vaccine development for humans.

Active and passive mechanical characterization of a human descending thoracic aorta with Klippel-Trenaunay syndrome

A human aorta from a female donor affected by Klippel-Trenaunay syndrome was retrieved during a surgery for organ donation for transplant. The aorta was preserved in refrigerated Belzer UW organ preservation solution and tested within a few hours for mechanical characterization with and without vascular smooth muscle activation. KCl and Noradrenaline were used as vasoactive agents in bubbled Krebs-Henseleit buffer solution at 37 °C. A quasi-static and a dynamic mechanical characterization of the full wall and the three individual layers were carried out for strips taken in longitudinal and circumferential directions. The full wall in the descending portion of the aorta underwent mechanical tests with and without smooth muscle activation. Results were compared to data obtained from healthy aortas and show a reduced stiffness of the full wall in circumferential direction. Also, a significant reduction of the response to vasoactive agents in circumferential direction was observed, while the longitudinal response was similar to healthy cases.

Prevalence of red blood cell alloantibodies among blood donors in the French Military Blood Institute: A 10-year retrospective study.

Screening for red blood cell alloantibodies (RBC-Ab) is a critical step in ensuring blood transfusion safety performed by blood donation screening laboratories. We aim to evaluate the prevalence of the RBC-Ab among healthy blood donors. Antibody screening of serum of all voluntary blood donors was performed as a routine immune-haematological procedure by a solid-phase method on a fully automated immunohaematology analyser. Positive sera were further investigated to identify the specificity of RBC-Ab by a commercially available red cell panel. Between January 2012 and December 2021, a total of 212,218 donations were screened for the presence of RBC-Ab, 74% from male donors (n = 157,898) and 26% from female donors (n = 54,320). Mean age at donation time was 32 ± 12 years. A total of 1007 donations were screened positive (0.47%), and 131 were confirmed positive for alloantibodies in their serum, yielding a prevalence of 0.06% (95% confidence interval: 0.05-0.07). Most frequent alloantibodies identified were of RH blood group system (64%), followed by anti-MNS (19%), anti-Kidd and Lewis (6% each) and anti-KEL (4%). The results showed a statistically higher prevalence of alloantibodies in women than men. Our results showed a lower prevalence as compared to the available data, which might be related to our study population. The prevalence of positive antibody screening in healthy donors in this study was found to be 0.47%, while the prevalence of alloantibodies was 0.06%. The most common alloantibodies were anti-RH1 (25%) and anti-RH3 (24%).

Transcriptional impact of EGFR activation in human female vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) are critical for the vascular tone, but they can also drive the development of vascular diseases when they lose their contractile phenotype and de-differentiate. Previous studies showed that the epidermal growth factor receptor (EGFR) of VSMC is critical for vascular health, but most of the underlying mechanisms by which VSMC-EGFR controls vascular fate have remained unknown. We combined RNA-sequencing and bioinformatics analysis to characterize the effect of EGFR-activation on the transcriptome of human primary VSMC (from different female donors) and to identify potentially affected cellular processes. Our results indicate that the activation of human VSMC-EGFR is sufficient to trigger a phenotypical switch toward a proliferative and inflammatory phenotype. The extent of this effect is nonetheless partly donor-dependent. Our hypothesis-generating study thus provides a first insight into mechanisms that could partly explain variable susceptibilities to vascular diseases in between individuals.

Transcriptome analysis reveals involvement of thiopurine S-methyltransferase in oxidation-reduction processes

Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in the deactivation of thiopurines and represents a major determinant of thiopurine-related toxicities. Despite its well-known importance in thiopurine metabolism, the understanding of its endogenous role is lacking. In the present study, we aimed to gain insight into the molecular processes involving TPMT by applying a data fusion approach to analyze whole-genome expression data. The RNA profiling was done on whole blood samples from 1017 adult male and female donors to the Estonian biobank using Illumina HTv3 arrays. Our results suggest that TPMT is closely related to genes involved in oxidoreductive processes. The in vitro experiments on different cell models confirmed that TPMT influences redox capacity of the cell by altering S-adenosylmethionine (SAM) consumption and consequently glutathione (GSH) synthesis. Furthermore, by comparing gene networks of subgroups of individuals, we identified genes, which could have a role in regulating TPMT activity. The biological relevance of identified genes and pathways will have to be further evaluated in molecular studies.

Genetic typing and intrafamilial transmission of human T-lymphotropic virus type 1 in non-endemic areas of China.

The origin and intrafamilial transmission of Human T-Lymphotropic Virus Type 1 (HTLV-1) in non-endemic populations such as China is still unknown. In this study, donors from blood banks/centers in China (including 28 provinces and Shenzhen city) during 2019 and 2021 were screened for HTLV-1/2 antibody, and all the reactive samples were tested using a line immunoassay (LIA) and quantitative polymerase chain reaction (qPCR). Samples that can be detected using qPCR were amplified and sequenced for the long terminal repeat (LTR) region. The positive donors were contacted to identify their relatives. As a result, 4,451,883 blood donors were totally tested, and 50 of them were confirmed to be HTLV-1/2 positive. Viral LTR sequences genotyped from 26 HTLV-1 carriers demonstrated that all had the HTLV-1a genotype, of which Transcontinental and Japanese subgroups accounted for half each. There were 17 family members of 11 index donors detected, and the HTLV-1 infection rate in the spouses of male index donors (83.3%, 5/6) was significantly higher than that in the husbands of female index donors (0.0%, 0/4). However, 7 children of HTLV-1 positive women were tested and found negative. Therefore, our findings indicated that HTLV-1 is spreading silently from high-endemic to low-endemic areas in China. To prevent further HTLV-1/2 transmission, an efficient HTLV-1/2 screening strategy and counseling of the virus carriers are essential.

Cell Count and Cell Density Decrease as Age Increases in Cadaveric Pediatric Medial Menisci

To examine the histologic changes in terms of cellularity, cell density, and nuclear shape in medial meniscal cellularity during skeletal development using pediatric cadaver specimens. Medial menisci from 26 pediatric cadavers, 11 female and 15 male (total 36 menisci), were obtained from tissue bank. Mean age of female donors was 34 months (1-108 months) and of male donors was 52 months (1-132 months). Menisci were processed and embedded in paraffin blocks. Each tissue block containing 6 representative areas of meniscus (anterior root, anterior horn, body [n= 2], posterior horn, and posterior root) was sectioned at 4 microns and stained with hematoxylin and eosin for evaluation of chondrocyte nuclei. Each of the 6 representative areas was imaged at 10×; one image on peripheral one-third of section, the second image on central two-thirds of the section. FIJI imaging software was used to measure cell count, cell density, and nuclear morphology (1= perfect circle). Data analysis included linear mixed models, Type II analysis of variance tests, and pairwise tests with the Tukey correction to assess statistical significance. Peripheral meniscus was more cellular than central meniscus. The cell count was found to decrease by 14% per year of age. Peripheral cell count decreased at a rate similar to the cell count in the central meniscus. Meniscal cell density was 2× higher peripherally than centrally. Overall average cell density in all locations in the menisci decreased by an average of 14% per year of age. The results of this study reveal decreases in cell count, cell density, and circularity as age increases in cadaveric pediatric medial menisci. To better understand the development of pediatric menisci at a cellular level and use this knowledge in the future on how to maintain the menisci in a younger, healthier state.

Hybrid and SARS-CoV-2-vaccine immunity in kidney transplant recipients

Kidney transplant recipients (KTR) are at high risk for severe COVID-19 and have demonstrated poor response to vaccination, making it unclear whether successive vaccinations offer immunity and protection. We conducted a serologically guided interventional study where KTR patients that failed to seroconvert were revaccinated and also monitored seroconversion of KTR following the Norwegian vaccination program. We analysed IgG anti-RBD Spike responses from dose 2 (n=432) up to after the 6th (n=37) mRNA vaccine dose. The frequency and phenotype of Spike-specific T and B cell responses were assessed in the interventional cohort after 3-4 vaccine doses (n=30). Additionally, we evaluated the Specific T and B cell response to breakthrough infection (n=32), measured inflammatory cytokines and broadly cross-neutralizing antibodies, and defined the incidence of COVID-19-related hospitalizations and deaths. The Norwegian KTR cohort has a male dominance (2323 males, 1297 females), PBMC were collected from 114 male and 78 female donors. After vaccine dose 3, most KTR developed Spike-specific T cell responses but had significantly reduced Spike-binding B cells and few memory cells. The B cell response included a cross-reactive subset that could bind Omicron VOC, which expanded after breakthrough infection (BTI) and gave rise to a memory IgG+B cell response. After BTI, KTR had increased Spike-specific T cells, emergent non-Spike T and B cell responses, and a systemic inflammatory signature. Late seroconversion occurred after doses 5-6, but 38% (14/37) of KTR had no detectable immunity even after multiple vaccine doses. Boosting vaccination can induce Spike-specific immunity that may expand in breakthrough infections highlighting the benefit of vaccination to protect this vulnerable population. CEPI and internal funds.

Impact of donor marrow cellularity on outcome of allogeneic stem cell transplantation

BackgroundOutcomes of allogeneic hematopoietic stem cell transplant (HSCT) are affected by a number of donor and patient related factors like extent of human leucocyte antigen mismatch, age, source of stem cells, female donor to male recipient and stem cell dose. All of these factors have been extensively investigated; however, effect of donor bone marrow cellularity on HSCT outcomes has not been evaluated. MethodsThis was a prospective study carried out at Armed forces bone marrow transplant center (AFBMTC/NIBMT) from January 2018 to December 2022. Bone marrow cellularity of donors was determined by separate assessment by two experienced Hematopathologists and classified as normocellular, hypocellular or hypercellular according to donor age. Total nucleated cells (TNC) were assessed by automated hematology analyzer and stem cell quantification was done by flowcytometric assay based on CD34, CD45 and 7-AAD immunophenotyping markers. Primary outcome measures were time to achieve neutrophil and platelet engraftment. Secondary outcome measures assessed were overall survival (OS), disease free survival (DFS) and graft versus host disease (GVHD). Frequency and percentage were calculated for categorical variables while Chi-square test was used for quantitative variables. Multivariate Cox regression analysis was used to determine significance of different variables and effect of cellularity on them. Kaplan Meier estimates with group differences were calculated using log rank tests for OS and DFS. A p value of 0.05 or less was considered statistically significant. ResultsCellularity of 95 donors was assessed, 39 (41.1 %) had normocellular marrow while 56 (58.9 %) were hypocellular for age. Median time from diagnosis to transplant was 13 months. In 38 (40 %) the indication for donation was for patients with bone marrow failure syndromes, 23 (24.2 %) for hematological malignancies, 21(22.1 %) for beta thalassemia major and 13 (13.6 %) for miscellaneous disorders including immune deficiencies. Median stem cell / CD 34 dose was 6 × 106/kg and median TNC dose was 5.09 × 108/kg. Median time of neutrophil engraftment was 13.6 days while that of platelet engraftment was 27.1 days. Mean OS was 84.2 %. OS for normocellular donors was 84.6 % and that for hypocellular donors was 83.9 % (p 0.995). DFS for normocellular donors was 84.6 % and for hypocellular donors was 83.9 % (p 0.96). No statistically significant association between the disease group and transplant type with donor marrow cellularity (p value 0.32 and 0.358 respectively) was determined. Multivariate logistic regression model and Backwald test showed no significant association between donor marrow cellularity and CD 34 dose (p 0.65), TNC (p 0.78), neutrophil engraftment (p 0.23), platelet engraftment (p 0.27), Acute GVHD (p 0.83), and chronic GVHD (p 0.44). ConclusionMajority of the donors had hypocellular bone marrow. Bone marrow cellularity had no statistically significant impact on the CD34 and TNC doses obtained; neither did it affect post-transplant neutrophil and platelet engraftment, OS, and DFS. Further population-based studies are required to confirm normal marrow cellularity in Pakistani population and the effects of various genetic and environment factors that make it hypocellular.

High Hepatitis E Virus (HEV) Seroprevalence and No Evidence of HEV Viraemia in Vietnamese Blood Donors.

The prevalence of hepatitis E virus (HEV) in the Vietnamese population remains underestimated. The aim of the present study was to investigate the seroprevalence of HEV IgG/IgM antibodies and the presence of HEV RNA in blood donors as a part of epidemiological surveillance for transfusion-transmitted viruses. Serum samples from blood donors (n = 553) were analysed for markers of past (anti-HEV IgG) and recent/ongoing (anti-HEV IgM) HEV infections. In addition, all serum samples were subsequently tested for HEV RNA positivity. The overall prevalence of anti-HEV IgG was 26.8% (n = 148/553), while the seroprevalence of anti-HEV IgM was 0.5% (n = 3/553). Anti-HEV IgG seroprevalence in male and female donors was similar (27.1% and 25.5%, respectively). A higher risk of hepatitis E exposure was observed with increasing age. None of the blood donors were HEV RNA positive, and there was no evidence of HEV viraemia. Although the absence of HEV viraemia in blood donors from Northern Vietnam is encouraging, further epidemiological surveillance in other geographical regions is warranted to rule out transfusion-transmitted HEV.

FRI378 Estetrol Prolongs Anagen In Healthy Female Scalp Hair Follicles By Positively Modulating Dermal Papilla Functions And Generation Of Progenitor Stem Cells Ex Vivo

Abstract Disclosure: C. Gerard: None. A. La Riche: None. S. Altendorf: None. V. Dion: None. L. Epping: None. T. Bíro: None. G. Dixon: None. M. Bertolini: None. The human hair follicle (HF) is a neuroendocrine mini-organ that responds to, but also generates, a wide range of hormones, which affect hair cycle and HF biology. HF functions are profoundly regulated by several intrafollicular neuroendocrine axes like the HF-equivalent of the hypothalamic-pituitary-adrenal and thyroid axes, as well as by local prolactin secretion. In this regard, estrogens exert a direct role in hair cycle regulation, since estrogen receptors are prominently expressed in the epithelium of scalp HFs. It has been shown that postmenopausal women increasingly experience female pattern hair loss (FPHL) due to the decrease in ovarian estrogen secretion. Conversely, elevated estrogen levels were found to correlate with enhanced numbers of anagen HFs during pregnancy. Estetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. E4 was recently approved as the estrogenic component of a new combined oral contraceptive and is currently in the late stage of clinical development for its use as a menopausal hormone therapy. In this study, we aimed to assess the capacity of E4 to regulate hair growth.Frontotemporal, full-length microdissected HFs from 4 healthy female donors (&amp;gt; 50 years) were exposed to different concentrations of E4 (300nM, 3µM and 30µM) ex vivo. Subsequently, hair cycle staging, hair matrix keratinocyte proliferation (Ki-67), dermal papilla (DP) inductivity (versican expression and alkaline phosphatase activity), fibroblast emigration (cell density in the DP, the dermal cup (DC) and the DP stalk), and the percentage of HFs stem cell (HFSC) progenitors (CD34+) was assessed by quantitative (immuno-)histomorphometry. E4 treatment led to a tendential (300nM and 30µM) or significant (3µM) increase in the number of anagen (growing) HFs, further supported by enhanced hair matrix keratinocyte proliferation. Although neither size nor cell density of DP was affected by the treatment with E4, a significant lower number of cells was detected in the DP stalk and DC, indicating less DP fibroblast emigration. Additionally, E4 treatment stimulated DP fibroblast inductivity as evidenced by a tendential up-regulation of versican expression and alkaline phosphatase activity in the DP, the latter even significantly in only anagen HFs. Moreover, application of 3µM E4 positively affected the generation of stem cell progeny by significantly increasing the percentage of CD34+ cells in the suprabulbar outer root sheath. Taken together, our data suggest that treatment with E4 boosts anagen maintenance, reduces DP fibroblast emigration, stimulates DP inductivity and promotes the expansion of HFSC progeny. Thus, these findings point at a potential role of E4 to prevent catagen development and changes known to be involved in HF miniaturization, and further encourage the exploration of E4 as possible treatment strategy for hair loss disorders such as FPHL. Presentation: Friday, June 16, 2023

Visible body, invisible care: Family, gender politics, and the female caregiver in living donor liver transplantation in Taiwan

This study contributes to existing research on gender inequities in living donor liver transplantation (LDLT) by employing a feminist and sociocultural theoretical perspective. Through participant observation and in-depth interviews with 36 participants from 13 families, including 13 donors, 10 recipients, and 13 caregivers, at the Chang Gung Memorial Hospital, this study provides a nuanced understanding of the complexities of family dynamics and gender relations throughout the decision-making and meaning-making processes for LDLT in Taiwan. The interviews were transcribed verbatim, translated from Mandarin to English, and analysed using grounded theory and thematic analysis. The findings reveal that LDLT is a gendered experience embodied through four aspects: 1) the impact of family dynamics and gender politics on decision-making, 2) societal and cultural perceptions of women's bodies and families, 3) the multiple roles and multifaceted care work of female caregivers, and 4) gender differences in the discourse on the meaning-making of LDLT. The study illuminates the complex interplay of family, gender, and care within the sociocultural landscape of Taiwan. It underscores the gendered and hierarchical division of care work within households and the vulnerability experienced by female caregivers and donors, as well as other disadvantaged family members. Furthermore, the study offers insights for healthcare professionals and policymakers to recognise the value of caregiving and the need for more support and resources for caregivers. Future research could adopt a multifaceted approach and inform interventions and policies aimed at reducing gender inequities in LDLT, moving beyond the narrow focus on numerical gender imbalance.

Age-at-death estimation based on micro-CT assessment of pubic symphysis: Potentially new methodological approach

BackgroundAlthough various methods for age-at-death estimation of skeletal remains are available, this is still an unsolved issue in forensic anthropology, especially concerning elderly individuals. Moreover, the lack of population-specific methods often made age-at-death estimation unreliable in other populations. AimOur study aimed to examine whether micro-computed tomography (micro-CT) analysis of pubic bone samples obtained from the contemporary Serbian population could be used in anthropological and forensic practice for age-at-death estimation. MethodologyThis study encompassed 62 pubic samples obtained from 26 adult male and 36 adult female cadaveric donors (age range: 22–91 years). Initially, staging according to the Suchey–Brooks phases was performed by two experienced investigators, followed by micro-CT assessment of pubic bone trabecular and cortical compartments (spatial resolution of the scans was 10 µm). ResultsOur results revealed an age-associated decline in trabecular and cortical micro-architecture of elderly male and female individuals, with the most prominent changes present in trabecular bone volume fraction and total porosity of the anterior and posterior cortical surface of the pubic bone. Those parameters were used to generate age-at-death estimation equations. One sample t-test did not reveal a significant difference between estimated age-at-death and real (known) age-at-death in the overall sample (mean absolute error [MAE] of 4.76 years), female (MAE of 9.66 years) and male cadaveric donors (MAE of 6.10 years, p > 0.05). ConclusionOur data indicated that micro-architectural features of trabecular and cortical compartments of pubic bone could potentially be applied as an additional reliable method for age-at-death estimation in the Serbian population.

Relationship between Iron Metabolic Parameters and Platelet Counts in Blood Donors

To investigate the correlation of iron metabolic parameters with platelet counts in blood donors. A total of 400 blood donors who met requirements of apheresis platelet donation were collected, and their hematological parameters were analyzed. The donors were divided into low ferritin group and normal group, the differences of hematological parameters between the two groups were compared, and the correlation of iron metabolic parameters and routine hematology parameters with platelet counts were analyzed. Whether male or female, low ferritin group had higher platelet counts than normal group (P < 0.01). Among the iron metabolic parameters, the platelet counts was negatively correlated with serum ferritin (SF), serum iron (SI), and transferrin saturation (TSAT) (r =-0.162, r =-0.153, r =-0.256), and positively correlated with total iron binding capacity (TIBC) and unsaturated iron binding capacity (UIBC) (r =0.219, r =0.294) in female blood donors. Platelet counts was also negatively correlated with SF, SI and TSAT (r =-0.188, r =-0.148, r =-0.224) and positively correlated with UIBC (r =0.220) in male blood donors. Among the routine hematology parameters, platelet counts was negatively correlated with mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and reticulocyte hemoglobin equivalent (Ret-He) in female blood donors (r =-0.236, r =-0.267, r =-0.213, r =-0.284). Platelet counts was also negatively correlated with MCH, MCHC and Ret-He in male blood donors (r =-0.184, r =-0.221, r =-0.209). In blood donors with low C-reactive protein level, the lower the iron store capacity, the lower the iron utilization, and the platelet counts tends to rise.

In-Vitro Assessment of Platelets Survival in Stored Platelet Concentrates in Ile-Ife, Nigeria

Introduction: Platelets are fragments of megakaryocytes circulating in the blood and its concentrates are therapeutic in substantial bleeding disorders. Efforts to ensure adequate product quality are required due to their short lifespan and lack of robustness. A descriptive longitudinal laboratory-based study was adopted in this study. The study aimed at determining platelets survival in stored platelet concentrates and evaluating thromboxane A2 for platelets function. Materials and Methods: Platelet concentrates were prepared manually using buffy coat, where about 50ml of concentrates suspended in plasma were allowed to rotate and agitate continuously on platelet agitator at room temperature (20-240C). Aliquots of 4ml each was collected serially for 9 days (day 0 to day 8) from 10 different platelet concentrates collected from 7 male blood donors and 3 female blood donors with age (mean± SD: 36 ± 7.14 years), weight (mean± SD: 66.8 ± 6.01kg), height (mean± SD: 163 ± 4.57cm). The samples were analyzed for platelets count, platelet distribution width (PDW), mean platelet volume (MPV), platelet-large cell ratio (P-LCR), using Automated Haematology analyzer (Sysmex XP-300) and thromboxane A2 using standard ELISA technique. Data analysis was carried out using mean, standard deviation as descriptive statistics and logistic regression as inferential statistics; and p &lt;0.05 was considered as evidence of statistical significance. Results: Socio-demographic characteristics had no effect on all parameters estimated. There is variation in platelet count and platelet indices values in stored platelet concentrates compared with the baseline values and the survival of platelets in stored platelet concentrates was relatively stable till day 4 after preparation but depreciation surfaced from day 5 to day 8 compared to baseline values. The study also showed that the degree of deterioration of thromboxane A2 was highly significant at day 3 (p&lt;0.05) while the best duration of storage for platelet concentrates in the study area is the first 3 days, though storage up to day 5 is acceptable (p&gt;0.05). Conclusion: This study confirms thromboxane A2 as a marker for platelet functionality. The best duration of storage for platelet concentrates in the study area is the first two (2) days when no significant deterioration was observed.

Imaging of the Microstructural Failure Mechanism in the Human Hip.

Imaging the bone microstructure under progressively increasing loads allows for observing the microstructural failure behavior of bone. Here, we describe a protocol for obtaining a sequence of three-dimensional microstructural images of the entire proximal femur under progressively increasing deformation, causing clinically relevant fractures of the femoral neck. The protocol is demonstrated using four femora from female donors aged 66-80 years at the lower end of bone mineral density in the population (T-score range = -2.09 to -4.75). A radio-transparent compressive stage was designed for loading the specimens replicating a one-leg stance, while recording the applied load duringmicro-computed tomography (micro-CT) imaging. The field of view was 146 mm wide and 132 mm high, and the isotropic pixel size was 0.03 mm. The force increment was based on finite-element predictions of the fracture load. The compressive stage was used to apply the displacement to the specimen and enact the prescribed force increments. Sub-capital fractures due to opening and shear of the femoral neck occurred after four to five load increments. The micro-CTimages and the reaction force measurements were processed to study the bone strain and energy absorption capacity. Instability of the cortex appeared at the early loading steps. The subchondral bone in the femoral head displayed large deformations reaching 16% beforefracture, and a progressive increase in the support capacity up tofracture. The deformation energy linearly increased with the displacement up tofracture, while the stiffness decreased to near-zero values immediately beforefracture. Three-fourths of the fracture energy was taken by the specimen during the final 25% force increment. In conclusion, the protocol developed revealed a remarkable energy absorption capacity, or damage tolerance, and a synergic interaction between the cortical and trabecular bone at an advanced donor age.

A-038 An Improved Multiplexed HTLC-HESI-MSMS Method for the Measurement of Total Testosterone in Serum

Abstract Background Testosterone is an anabolic steroid produced primarily in the testicles of males and mainly in the ovaries of females. Imbalance of testosterone is the primary cause of hypogonadism in males, hirsutism and virilization in females, osteoporosis, and diabetes mellitus. Accurate and fast-turnaround measurement of testosterone is critical for diagnosis, prevention, and treatment of testosterone-related diseases in adults and children. In a previous study, testosterone was derivatized with methoxyamine and hydroxylamine in a multiplexed high turbulence liquid chromatography with heated-electrospray ionization-tandem mass spectrometry (HTLC-HESI-MS/MS) method. Using these reagents, differential mass-tagging of testosterone allowed different specimens to be combined in a single injection to increase assay throughput. However, interference was observed for hydroxylamine-derivatized testosterone in serum collected in serum separator tubes (SST). In the current study, our objectives were to (1) replace hydroxylamine with a new derivatizing agent not subject to interference and (2) develop an improved HTLC-HESI-MS/MS method for multiplexed total testosterone measurement. Methods Healthy male and female donor specimens were collected in SST and red-top tubes. Serum was mixed with stable isotope-labeled testosterone as internal standard (ISTD) prior to protein precipitation. Testosterone in the supernatants was derivatized, enriched by solid phase extraction, dried, and reconstituted. Specimens were injected on the HTLC system for on-line extraction, transferred to analytical columns, and eluted using binary gradient. Testosterone derivatives and ISTDs were detected and analyzed on a Triple Quadrupole Mass Spectrometer (Thermo) equipped with HESI. Ionization and multiple reaction monitoring (MRM) scan parameters were optimized for maximized ion transmission and sensitive, specific, and stable quantitation. MRM transitions were used as quantifiers and qualifiers for both testosterone derivatives. Also assessed were ion suppression using T-column infusion, specimen stability by storage in SST tubes at 4 °C, isobaric interference from dehydroepiandrosterone (DHEA), and carryover. Results Intra- and inter-assay coefficients of variation (CV%) for total testosterone levels were &amp;lt;5% at 12, 80, 250, and 1200 ng/dL for both testosterone derivatives. Limits of quantitation (LOQs) for both derivatives were &amp;lt;1 ng/dL. Calibration linearity was verified across the measurement range of 2.5–2000 ng/dL. Both agents were equivalent in derivatizing and recovering testosterone from serum based on a comparison of their quantitative results (R = 0.991, bias = −3.8 ng/dL). No interference was observed in serum collected in SST. Serum specimens collected in SST and red-top tubes from same donors gave equivalent results (R = 0.991, bias = 0.2 ng/dL). Ion suppression was calculated as less than 20% for both derivatives. CV% of the ion-ratios of both derivatives were less than 15% and were stable across the linear range. Testosterone levels were stable in SST for 72 h (at 4 °C) with the serum not separated from the gel. No isobaric interference and no carry over were observed. Conclusion We have developed and validated an improved multiplexed HTLC-HESI-MS/MS testosterone assay using new derivatizing agents. The current method allowed accurate, rapid, specific, and stable analysis of serum total testosterone. SST is an acceptable specimen type for the current method. Precision, accuracy, linearity, ion suppression, and sample stability in SST were validated.

Outcomes of tricuspid valve regurgitation after heart transplantation; a single center retrospective cohort study

BackgroundTricuspid valve regurgitation (TR) of the donor's heart is a common problem that can develop immediately after cardiac transplantation. There is still little data about its impact on clinical outcomes. We aimed to evaluate the short-term and 1-year effects of significant TR after heart transplantation. We retrospectively analyzed 120 patients who underwent elective orthotopic heart transplantation between 2011 and 2022. According to the postoperative TR, the patients were divided into insignificant (n= 106) and significant (n= 14) TR groups.ResultsPatients with significant TR were younger [median age: 38 (Q1- Q3: 36- 39) vs. 49 (35- 55) years, p=0.037] and had a higher prevalence of nonischemic cardiomyopathy (85.71% vs. 42.45%, p=0.003) and donor female gender (35.7% vs. 19.8%,p=0.021). The patients with significant TR had a longer ICU stay [19 (14-27) vs. 11 (9-14) days, p=0.001], more frequent acute kidney injury (57.14% vs. 32.08%, p=0.023), and continuous renal replacement therapy (14.3% vs. 5.66%, p=0.031), with higher 30-day mortality (7.14% vs. 1.9%, p=0.046) and 1-year mortality (14.3% vs. 2.8%, p=0.022) compared to patients with insignificant TR. The degree of TR significantly decreased during the follow-up [β: -0.18 (95% CI: -0.25 to -0.10); p<0.001]. All patients with TR were managed medically without surgical interventions. Logistic multivariable regression revealed that nonischemic heart disease (OR: 3.41,95% CI: 1.31-41.2, p=0.024) and female donor (OR:1.56, 95%CI: 1.13-25.3, p=0.013) independently predicted significant postoperative TR.ConclusionsSignificant TR was associated with early mortality and morbidities with prolonged hospitalization. The degree of TR significantly decreased during the first year after transplantation.

Utilization of Paragonix Sherpapak Cardiac Transport System for the Preservation of Donor Hearts After Circulatory Death

Donation after circulatory death (DCD) heart transplantation is being increasingly adopted by transplant centers. The optimal method of DCD heart preservation during transport after in situ thoracoabdominal normothermic regional perfusion (TA-NRP) is not known. We evaluated our experience with the Paragonix SherpaPak Cardiac Transport System (SCTS) for the transport of DCD cardiac allografts after TA-NRP recovery between January 2021 and December 2022. We collected and evaluated donor characteristics, allograft ischemic intervals, and recipient baseline demographic and clinical variables, and short-term outcomes. Twelve recipients received DCD grafts recovered with TA-NRP and transported in SCTS during the study period. The median age of 10 male and 2 female donors was 32 years (min 15, max 38). The median duration of functional warm ischemia was 12 minutes (min 8, max 22). Hearts were preserved in SCTS for a median of 158 minutes (min 37, max 224). Median recipient age was 61 years (min 28, max 70). Ten recipients (83%) survived to hospital discharge, with one death attributable to graft dysfunction (8%). The median vasoactive-inotropic (VIS) score at 72 hours post-transplantation of the entire cohort was 6 (min 0, max 15). The median length of intensive care unit stay in hospital survivors was 5 days (min 3, max 17) days and hospital stay 17 days (min 9, max 37). The Paragonix SCTS provides efficacious preservation of DCD grafts for ≥3.5 hours. Organs transported with this device showed satisfactory post-transplantation function.

Selective effects of estradiol on human corneal endothelial cells

In Fuchs endothelial corneal dystrophy (FECD), mitochondrial and oxidative stresses in corneal endothelial cells (HCEnCs) contribute to cell demise and disease progression. FECD is more common in women than men, but the basis for this observation is poorly understood. To understand the sex disparity in FECD prevalence, we studied the effects of the sex hormone 17-β estradiol (E2) on growth, oxidative stress, and metabolism in primary cultures of HCEnCs grown under physiologic ([O2]2.5) and hyperoxic ([O2]A) conditions. We hypothesized that E2 would counter the damage of oxidative stress generated at [O2]A. HCEnCs were treated with or without E2 (10 nM) for 7–10 days under both conditions. Treatment with E2 did not significantly alter HCEnC density, viability, ROS levels, oxidative DNA damage, oxygen consumption rates, or extracellular acidification rates in either condition. E2 disrupted mitochondrial morphology in HCEnCs solely from female donors in the [O2]A condition. ATP levels were significantly higher at [O2]2.5 than at [O2]A in HCEnCs from female donors only, but were not affected by E2. Our findings demonstrate the resilience of HCEnCs against hyperoxic stress. The effects of hyperoxia and E2 on HCEnCs from female donors suggest cell sex-specific mechanisms of toxicity and hormonal influences.