A simplified CD34+ based preharvest prediction tool for HPC(A) collection.

BACKGROUND: Hematopoietic stem cell transplantation is an important treatment that is dependent on the collection of sufficient CD34+ hematopoietic progenitor cells. The peripheral blood CD34 count (PB CD34+ counts) measured by flow cytometry can be used in predicting CD34+ stem cell yields hours before the completion of collection. Previously described formulas to predict the yield have used many different variables. As such, there is currently no consensus on an industry-standard algorithm or formula. STUDY DESIGN AND METHODS: Retrospective reviews of same-day PB CD34+ counts and the ensuing absolute CD34+ yields of mobilized donors (allogeneic and autologous) were used to develop and validate a formula using regression analysis to predict the CD34+ stem cell yield. A metric of prediction correlation, using root mean square error (RMSE), was used to assess the robustness of our prediction formula in addition to comparisons with two other published formulas, as well as subset analysis. RESULTS: A formula in the form of y = mxb with r = 0.95 and 95% confidence intervals was generated and validated. The ratio of actual to predicted yield demonstrated a high correlation coefficient (r = 0.96) with linear regression and overall RMSE of 228.4, which was lower than the two prior studies (calculated RMSE = 330.8 and 405.2). Subset analyses indicated male patients, lymphoma patients, and patients >60 years of age demonstrated lower RMSEs. CONCLUSION: We have demonstrated a simple yet robust formula that can be used prospectively to accurately predict the CD34+ stem cell yield in both autologous and allogeneic donors, which also accounts for recipient weight.

Identifying correlations between donor demographics and isohemagglutinin titers as a potential method to screen for low-titer group O whole blood.

BACKGROUND: With more hospitals using low-titer group O whole blood in trauma resuscitation, having an efficient screening method for low-titer donors is critical. Our blood center uses an automated screen for high-titer isohemagglutinins in our platelet donations while collecting detailed donor demographic information. Using this data, we can identify key demographics often associated with titer status, thereby helping develop a donor-triaging method for titering. STUDY DESIGN AND METHODS: Titer results were read with an automated microplate system as either high or low, based on agglutination, with a cutoff equivalent to 1:256 (both anti-A and anti-B). Donor demographic data analyzed included date of donation, blood group, age, gender, and ethnicity. RESULTS: 57,508 donations were collected from 2073 unique donors between 2014 and 2018. We found the following demographics to be correlated with titer status: gender, ABO blood group, age, and ethnicity. Variability in titer status was identified in 215 individuals. This represented around 10 % of the total unique donors and was split equally amongst gender. We also found that donors between the ages of 41-60 ha d the highest likelihood of having variability in titer status, peaking at 13 %, and this proportion declined past age 60. CONCLUSION: Titer status is associated with the following donor demographics: gender, ABO type, age, and ethnicity. We also discovered that variability in titer status is correlated with age. In blood centers that do not have automated and routine titer screening procedure, these findings could be used as a method to efficiently identify low-titer donors a-priori.

Stability of anti-A blood group titers among blood group B renal transplant candidates.

BACKGROUND: As deceased donor kidney allocation is based in part on blood type compatibility, group B candidates are disadvantaged due to their disproportionate representation on the wait list compared to the group B donor pool. To mitigate this discrepancy, group B candidates can receive group A2 or A2 B donor kidneys if their anti-A titers are below a predetermined cutoff. Currently, eligibility is reverified quarterly to UNet based on individual center protocols, which can vary due to a lack of set guidelines for monitoring ABO titers in these patients. Our goal was to assess the stability of anti-A titers in blood group B renal transplant candidates over time to provide data that could aid in the development of standardized ABO titer protocols. STUDY DESIGN AND METHODS: Titers performed between January 2011 and December 2015 were assessed for 191 group B patients with two or more documented titers. RESULTS: Fifty patients (26%) were ineligible, as the first titer exceeded the cutoff of 8. Of the remaining 141 patients, 19 (13%) became ineligible as the second titer exceeded 8. Thirty-nine patients (28%) had no change in titer between samples, while 71 (50%) had a titer change that never exceeded 8. Only 12 patients (8.5% of total) experienced a titer change that affected eligibility after the second test. CONCLUSION: Although patients experience some variability in anti-A titers over time, in most cases, stability did not affect candidate eligibility. Our results indicate that regular testing beyond the second titer may be unnecessary and represent test overutilization.

The UPF3B gene, implicated in intellectual disability, autism, ADHD and childhood onset schizophrenia regulates neural progenitor cell behaviour and neuronal outgrowth.

Loss-of-function mutations in UPF3B result in variable clinical presentations including intellectual disability (ID, syndromic and non-syndromic), autism, childhood onset schizophrenia and attention deficit hyperactivity disorder. UPF3B is a core member of the nonsense-mediated mRNA decay (NMD) pathway that functions to rapidly degrade transcripts with premature termination codons (PTCs). Traditionally identified in thousands of human diseases, PTCs were recently also found to be part of 'normal' genetic variation in human populations. Furthermore, many human transcripts have naturally occurring regulatory features compatible with 'endogenous' PTCs strongly suggesting roles of NMD beyond PTC mRNA control. In this study, we investigated the role of Upf3b and NMD in neural cells. We provide evidence that suggests Upf3b-dependent NMD (Upf3b-NMD) is regulated at multiple levels during development including regulation of expression and sub-cellular localization of Upf3b. Furthermore, complementary expression of Upf3b, Upf3a and Stau1 stratify the developing dorsal telencephalon, suggesting that alternative NMD, and the related Staufen1-mediated mRNA decay (SMD) pathways are differentially employed. A loss of Upf3b-NMD in neural progenitor cells (NPCs) resulted in the expansion of cell numbers at the expense of their differentiation. In primary hippocampal neurons, loss of Upf3b-NMD resulted in subtle neurite growth effects. Our data suggest that the cellular consequences of loss of Upf3b-NMD can be explained in-part by changes in expression of key NMD-feature containing transcripts, which are commonly deregulated also in patients with UPF3B mutations. Our research identifies novel pathological mechanisms of UPF3B mutations and at least partly explains the clinical phenotype of UPF3B patients.

Drosophila orthologue of WWOX, the chromosomal fragile site FRA16D tumour suppressor gene, functions in aerobic metabolism and regulates reactive oxygen species.

Common chromosomal fragile sites FRA3B and FRA16D are frequent sites of DNA instability in cancer, but their contribution to cancer cell biology is not yet understood. Genes that span these sites (FHIT and WWOX, respectively) are often perturbed (either increased or decreased) in cancer cells and both are able to suppress tumour growth. While WWOX has some tumour suppressor characteristics, its normal role and functional contribution to cancer has not been fully determined. We find that a significant proportion of Drosophila Wwox interactors identified by proteomics and microarray analyses have roles in aerobic metabolism. Functional relationships between Wwox and either CG6439/isocitrate dehydrogenase (Idh) or Cu-Zn superoxide dismutase (Sod) were confirmed by genetic interactions. In addition, altered levels of Wwox resulted in altered levels of endogenous reactive oxygen species. Wwox (like FHIT) contributes to pathways involving aerobic metabolism and oxidative stress, providing an explanation for the 'non-classical tumour suppressor' behaviour of WWOX. Fragile sites, and the genes that span them, are therefore part of a protective response mechanism to oxidative stress and likely contributors to the differences seen in aerobic glycolysis (Warburg effect) in cancer cells.

Conservation and expression of PIWI-interacting RNA pathway genes in male and female adult gonad of amniotes.

The PIWI-interacting RNA (piRNA) pathway is essential for germline development and transposable element repression. Key elements of this pathway are members of the piRNA-binding PIWI/Argonaute protein family and associated factors (e.g., VASA, MAELSTROM, and TUDOR domain proteins). PIWI-interacting RNAs have been identified in mouse testis and oocytes, but information about the expression of the different piRNA pathway genes, in particular in the mammalian ovary, remains incomplete. We investigated the evolution and expression of piRNA pathway genes in gonads of amniote species (chicken, platypus, and mouse). Database searches confirm a high level of conservation and revealed lineage-specific gain and loss of Piwi genes in vertebrates. Expression analysis in mammals shows that orthologs of Piwi-like (Piwil) genes, Mael (Maelstrom), Mvh (mouse vasa homolog), and Tdrd1 (Tudor domain-containing protein 1) are expressed in platypus adult testis. In contrast to mouse, Piwil4 is expressed in platypus and human adult testis. We found evidence for Mael and Piwil2 expression in mouse Sertoli cells. Importantly, we show mRNA expression of Piwil2, Piwil4, and Mael in oocytes and supporting cells of human, mouse, and platypus ovary. We found no Piwil1 expression in mouse and chicken ovary. The conservation of gene expression in somatic parts of the gonad and germ cells of species that diverged over 800 million yr ago indicates an important role in adult male and female gonad.

Biometric Identification System as a Potential Source of Nosocomial Infection Among Medical Students.

In the current situation, biometric devices, electronic devices, and medical apparatus usage cannot be restricted in hospitals and offices. These devices will act as a vehicle for the transmission of bacterial agents. Our study observed that a biometric device is acting as a vehicle/source of spreading bacterial agents. It will be more in the hospital environment. The only way to protect from infections is strict adherence to infection control and good hygienic practices. We also observed fewer samples containing yielding of bacteria, indicating that either student is not interacting with biometric devices or is more aware of infection spread and followed the hand hygiene practice with alcohol.

Universal Engraftment after Allogeneic Hematopoietic Cell Transplantation Using Cryopreserved CD34-Selected Grafts.

As a result of the COVID-19 pandemic, most centers performing allogeneic hematopoietic cell transplantation (allo-HCT) have switched to the use of cryopreserved grafts. Previous investigators have suggested that cryopreserved allografts may heighten risk of nonengraftment. To date, no study has investigated the effect of cryopreservation of CD34-selected hematopoietic progenitor cells (CD34+ HPCs) used as the sole graft source. In this study, we sought to evaluate outcomes after unrelated donor or matched sibling allo-HCT with cryopreserved CD34+ HPCs. This was a single-center analysis of adult patients with hematologic malignancies who underwent allo-HCT with cryopreserved CD34-selected allo-HCT grafts between January 2010 and June 2017. All patients received ablative conditioning and antirejection prophylaxis with rabbit antithymocyte globulin. G-CSF-mobilized leukapheresis products underwent CD34 selection using the CliniMACS Reagent System. Cells were then cryopreserved in DMSO (final concentration 7.5%) to -90 °C using a controlled-rate freezing system before being transferred to vapor-phase liquid nitrogen storage. In internal validation, this method has shown 92% mean CD34+ cell viability and 99.7% mean CD34+ cell recovery. Engraftment was defined as the first of 3 consecutive days of an absolute neutrophil count of ≥0.5. Platelet recovery was recorded as the first of 7 consecutive days with a platelet count ≥20 K/µL without transfusion. Kaplan-Meier methodology was used to estimate overall survival (OS) and relapse-free survival (RFS), and cumulative incidence functions were used to estimate rates of relapse, nonrelapse mortality (NRM), and acute graft-versus-host disease (GVHD). A total of 64 patients received a cryopreserved CD34-selected graft. The median CD34+ cell count before cryopreservation was 6.6 × 106/kg (range, 1.4 to 16.1 × 106/kg), and the median CD3+ cell count was 2.0 × 103/kg (range, 0 to 21.1 × 106/kg). All patients were engrafted, at a median of 11 days post-HCT (range, 8 to 14 days). One patient had poor graft function in the setting of cytomegalovirus viremia, necessitating a CD34-selected boost on day +57. The median time to platelet recovery was 16 days (range, 13 to 99 days). The estimated 2-year OS was 70% (95% confidence interval [CI], 58% to 83%) with cryopreserved grafts versus 62% (95% CI, 57% to 67%) with fresh grafts (hazard ratio [HR], 0.86; 95% CI, 0.54 to 1.35; P = .5). The estimated 2-year RFS in the 2 groups was 59% (95% CI, 48% to 74%) versus 56% (95% CI, 51% to 61%; HR, 1.01; 95% CI, 0.68 to 1.51; P > .9). The cumulative incidence of relapse at 2 years was 29% (95% CI, 17% to 41%) versus 23% (95% CI, 19% to 27%; P = .16), and the cumulative incidence of NRM at 2 years was 17% (95% CI, 9% to 28%) versus 23% (95% CI, 19% to 28%; P = .24). The cumulative incidence of grade II-IV acute GVHD by day +100 was 16% with cryopreserved grafts (95% CI, 8% to 26%) and 16% (95% CI, 13% to 20%; P = .97) with fresh grafts. Moderate to severe chronic GVHD by day +365 occurred in only 1 recipient of a cryopreserved graft (2%). Our data show that in patients with hematologic malignancies who received cryopreserved allogeneic CD34+ HPCs, engraftment, GVHD, and survival outcomes were consistent with those seen in recipients of fresh allogeneic CD34+ HPC grafts at our center. Our laboratory validation and clinical experience demonstrate the safety of our cryopreservation procedure for CD34-selected allografts.

Acute secretory cell toxicity and epithelial exfoliation after smoke inhalation injury in sheep: an electron and light microscopic study.

Smoke inhalation injury promotes exfoliation of the upper airway columnar epithelium. Tracheal tissues from sheep 30 min after smoke exposure show intact epithelial areas, areas of epithelial disruption with loss of columnar cells and areas denuded of columnar cells. In intact areas detaching ciliated cells can be seen raised above the apical surface. This study aims to assess cell-specific toxicity by examining intact epithelium after inhalation injury. The junctional adhesion integrity between columnar and basal cells and the type of cells initially being displaced were also studied using light (LM) and transmission electron microscopy (TEM). TEM assessment of intact areas of sheep tracheal tissue (n=3) 30 min after exposure showed secretory cell toxicity including extrusion of cytoplasmic contents. In cells with severe secretory cell cytoplasmic disruption, loss of desmosomal junctions between the secretory and adjacent ciliated cells was evident. The number of desmosomes visible between columnar cells and basal cells was reduced (2.8 ± 1.8) in smoke-exposed animals compared to those in uninjured animals (5.0 ± 2.7), p=0.008. Serial sections of intact regions found 52 cells being displaced from the epithelium. All detaching cells were identified as ciliated cells. These studies show that the acute effects of inhalation injury include selective secretory cell toxicity which is associated with loss of junctional adhesion mechanisms and displacement of ciliated cells. Improved understanding of acute hypersecretory responses and epithelial integrity after exposure to toxic agents may improve understanding of epithelial fragility in airway disease.

Substance P antagonist CP-96345 blocks lung vascular leakage and inflammation more effectively than its stereoisomer CP-96344 in a mouse model of smoke inhalation and burn injury.

The recently developed murine model of smoke inhalation and burn (SB) injury was used to study the effect of the substance-P antagonist CP96345. C57BL/6 mice were pre-treated with an i.v. dose of a specific NK-1 receptor antagonist, CP9635, or its inactive enantiomer, CP96344, (10 mg/Kg) 1 h prior to SB injury per protocol (n = 5). Mice were anesthetized and exposed to cooled cotton smoke, 2X 30 s, followed by a 40% total body surface area flame burn per protocol. At 48 h after SB injury Evans Blue (EB) dye and myeloperoxidase (MPO) were measured in lung after vascular perfusion. Lungs were also analyzed for hemoglobin (Hb) and wet/dry weight ratio. In the current study, CP96345 pre-treatment caused a significant decrease in wet/dry weight ratio (23%, p = 0.048), EB (31%, p = 0.047), Hb (46%, p = 0.002), and MPO (54%, p = 0.037) levels following SB injury compared to animals with SB injury alone. CP-96344 pre-treatment caused an insignificant decrease in wet/dry weight ratio (14%, p = 0.18), EB (16%, p = 0.134), Hb (9%, p = 0.39), and an insignificant increase in MPO (4%, p = 0.79) as compared to mice that received SB injury alone. As expected, levels of EB, Hb, MPO, and wet/dry weight ratios were all significantly (p < 0.05) increased 48 h following SB injury alone compared to respective sham animals. In conclusion, the current study indicates that pre-treatment with a specific NK-1R antagonist CP-96345 attenuates the lung injury and inflammation induced by SB injury in mice.

The impact of transfused blood products on deceased donor HLA typing.

Accurate deceased donor HLA typing assumes that the blood sample tested contains only DNA from the organ donor. Prior to procurement, many organ donors are transfused at least one unit of red blood cells (RBC). Non-organ donor DNA acquired from transfusions may result in incorrect and/or ambiguous HLA typing. To address this question, we investigated the impact of RBC transfusion on organ donor HLA typing by using different in vitro transfusion models: leukoreduced (LR) and non-LR RBCs. Various quantities of LR and non-LR RBCs were added to normal peripheral blood and HLA typing was performed by real time PCR. Our results show that HLA typing of deceased donors can be impacted dependent upon the type and quantity of transfused RBCs. Importantly, if LR RBCs are given, HLA typing is unlikely to be affected, precluding the need to delay typing and obtain an alternative source of donor DNA.

Chitosan wound dressing with hexagonal silver nanoparticles for hyperthermia and enhanced delivery of small molecules.

Chitosan films were synthesized with hexagonal silver nanoparticles (Ag NP). The unique shape and size of the Ag NP shift the optical absorption into the infrared. Stimulation of the nanoparticles with infrared light was used to generate heat and facilitate intracellular delivery of fluorescently-labeled dextran molecules. Chitosan films prepared with hexagonal or spherical Ag NP were characterized by optical and thermal analyses, and X-ray diffraction. There were found to be slight differences between how the chitosan molecular chains interface with the Ag NP depending upon shape of the nanoparticle. Viability of cells associated with dermal wound healing was evaluated on chitosan films prepared with hexagonal or spherical Ag NP, with both keratinocytes and fibroblasts having normal or moderately enhanced growth on films containing hexagonally-shaped nanoparticles.

TROP-2 expression in papillary thyroid carcinoma: Potential Diagnostic Utility.

TROP-2 is a type I transmembrane glycoprotein which is over-expressed in various malignancies, and is related to epithelial cell adhesion molecule (EpCAM), also called TROP-1, gp40, and KSA. In this study, we evaluated TROP-2 expression in papillary thyroid carcinoma (PTC) and compared it to other thyroid neoplastic and non-neoplastic lesions. Immunohistochemical (IHC) evaluation for TROP-2 was performed on 137 thyroid fine needle aspiration (FNA) cell blocks (CB) which included classic PTC (64), follicular variant PTC (FVPTC) (10), anaplastic thyroid carcinoma (AC) (2), medullary carcinoma (MC) (8), follicular neoplasms (FN) (8), Hurthle cell neoplasms (HCN) (9), follicular lesion of uncertain significance (FLUS) (12), and benign thyroid nodule (BTN) (24). IHC for TROP-2 expression was also performed on 331 BTN and malignant tumor tissue sections in tissue microarray (TMA). Membranous staining in >5% of tumor cells was considered positive. TROP-2 stained 61 of 64 PTC CB, 7 of 10 FVPTC CB, and 9 of 12 FLUS CB. All other cases were negative for TROP-2. TROP-2 showed a sensitivity of 95.31% and specificity of 89% for classic PTC in FNA CB. In TMA samples, TROP-2 stained 54 of 60 classic PTC cases and hence showed a high sensitivity and specificity. All BTN in CB and TMA were negative. We conclude that TROP-2 is a highly sensitive and specific IHC marker for identifying classic PTC. TROP-2 may play an important role in diagnosing classic PTC, especially in equivocal cases. This study also identifies a strong role for TROP-2 in separating PTC from BTN.