SARS-CoV-2 Mac1 is required for IFN antagonism and efficient virus replication in cell culture and in mice.

Several coronavirus (CoV) encoded proteins are being evaluated as targets for antiviral therapies for COVID-19. Included in these drug targets is the conserved macrodomain, or Mac1, an ADP-ribosylhydrolase and ADP-ribose binding protein encoded as a small domain at the N terminus of nonstructural protein 3. Utilizing point mutant recombinant viruses, Mac1 was shown to be critical for both murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV virulence. However, as a potential drug target, it is imperative to understand how a complete Mac1 deletion impacts the replication and pathogenesis of different CoVs. To this end, we created recombinant bacterial artificial chromosomes (BACs) containing complete Mac1 deletions (ΔMac1) in MHV, MERS-CoV, and SARS-CoV-2. While we were unable to recover infectious virus from MHV or MERS-CoV ΔMac1 BACs, SARS-CoV-2 ΔMac1 was readily recovered from BAC transfection, indicating a stark difference in the requirement for Mac1 between different CoVs. Furthermore, SARS-CoV-2 ΔMac1 replicated at or near wild-type levels in multiple cell lines susceptible to infection. However, in a mouse model of severe infection, ΔMac1 was quickly cleared causing minimal pathology without any morbidity. ΔMac1 SARS-CoV-2 induced increased levels of interferon (IFN) and IFN-stimulated gene expression in cell culture and mice, indicating that Mac1 blocks IFN responses which may contribute to its attenuation. ΔMac1 infection also led to a stark reduction in inflammatory monocytes and neutrophils. These results demonstrate that Mac1 only minimally impacts SARS-CoV-2 replication, unlike MHV and MERS-CoV, but is required for SARS-CoV-2 pathogenesis and is a unique antiviral drug target.

Mutation of a highly conserved isoleucine residue in loop 2 of several ß-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental for replication.

All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in non-structural protein 3. Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the glycine-isoleucine-phenylalanine motif. While we previously demonstrated the importance of the glycine residue for CoV replication and pathogenesis, the impact of the isoleucine and phenylalanine residues remains unknown. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that correlated with attenuated replication and pathogenesis of F-A mutant MERS-CoV and SARS-CoV-2 viruses in cell culture and mice. In contrast, the I-A mutant proteins had normal enzyme activity and enhanced ADP-ribose binding. Despite only demonstrating increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 viruses were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. IMPORTANCE: The conserved coronavirus (CoV) macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases and is critical for CoV replication and pathogenesis. As such, Mac1 is a potential therapeutic target for CoV-induced disease. However, we lack a basic knowledge of how several residues in its ADP-ribose binding pocket contribute to its biochemical and virological functions. We engineered mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and viruses for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Interestingly, a Mac1 isoleucine-to-alanine mutant protein had enhanced ADP-ribose binding which proved to be detrimental for virus replication, indicating that this isoleucine controls ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease.

PARP12 is required to repress the replication of a Mac1 mutant coronavirus in a cell- and tissue-specific manner.

ADP-ribosyltransferases (ARTs) mediate the transfer of ADP-ribose from NAD+ to protein or nucleic acid substrates. This modification can be removed by several different types of proteins, including macrodomains. Several ARTs, also known as PARPs, are stimulated by interferon indicating ADP-ribosylation is an important aspect of the innate immune response. All coronaviruses (CoVs) encode for a highly conserved macrodomain (Mac1) that is critical for CoVs to replicate and cause disease, indicating that ADP-ribosylation can effectively control coronavirus infection. Our siRNA screen indicated that PARP12 might inhibit the replication of a murine hepatitis virus (MHV) Mac1 mutant virus in bone-marrow-derived macrophages (BMDMs). To conclusively demonstrate that PARP12 is a key mediator of the antiviral response to CoVs both in cell culture and in vivo, we produced PARP12-/-mice and tested the ability of MHV A59 (hepatotropic/neurotropic) and JHM (neurotropic) Mac1 mutant viruses to replicate and cause disease in these mice. Notably, in the absence of PARP12, Mac1 mutant replication was increased in BMDMs and mice. In addition, liver pathology was also increased in A59-infected mice. However, the PARP12 knockout did not restore Mac1 mutant virus replication to WT virus levels in all cell or tissue types and did not significantly increase the lethality of Mac1 mutant viruses. These results demonstrate that while PARP12 inhibits MHV Mac1 mutant virus infection, additional PARPs or innate immune factors must contribute to the extreme attenuation of this virus in mice. IMPORTANCE Over the last decade, the importance of ADP-ribosyltransferases (ARTs), also known as PARPs, in the antiviral response has gained increased significance as several were shown to either restrict virus replication or impact innate immune responses. However, there are few studies showing ART-mediated inhibition of virus replication or pathogenesis in animal models. We found that the CoV macrodomain (Mac1) was required to prevent ART-mediated inhibition of virus replication in cell culture. Using knockout mice, we found that PARP12, an interferon-stimulated ART, was required to repress the replication of a Mac1 mutant CoV both in cell culture and in mice, demonstrating that PARP12 represses coronavirus replication. However, the deletion of PARP12 did not fully rescue Mac1 mutant virus replication or pathogenesis, indicating that multiple PARPs function to counter coronavirus infection.

Pre-operative anemia and peri-operative transfusion are associated with poor oncologic outcomes in cancers of the esophagus: potential impact of patient blood management on cancer outcomes.

BACKGROUND: Both Red Blood Cell (RBC) transfusion and anemia are thought to negatively impact cancer survival. These effects have been reported with mixed findings in cancer of the esophagus. The potential impact of the application of restrictive transfusion strategies on this patient population has not been defined. MATERIALS AND METHODS: We conducted a retrospective study of esophagectomies and studied cases based on whether they were anemic or were transfused peri-operatively. Clinical characteristics and known clinicopathologic prognosticators were compared between these groups. Survival was compared by Cox proportional hazard modeling. Post-operative transfusions were assessed for compliance with restrictive transfusion thresholds. RESULTS: Three-hundred ninety-nine esophagectomy cases were reviewed and after exclusions 348 cases were analyzed. The median length of follow-up was 33 months (range 1-152 months). Sixty-four percent of patients were anemic pre-operatively and 22% were transfused. Transfusion and anemia were closely related to each other. Microcytic anemia was uncommon but was evaluated and treated in only 50% of cases. Most anemic patients had normocytic RBC parameters. Transfusion but not anemia was associated with a protracted/prolonged post-operative stay. Transfusion and anemia were both associated with reduced survival however only anemia was associated with decreased survival in multi-variable modeling. Sixty-eight percent of patients were transfused post-operatively and 11% were compliant with the restrictive threshold of 7 g/dL. CONCLUSIONS: Pre-operative anemia and transfusion are closely associated, however only anemia was found to compromise survival in our esophageal cancer cohort, supporting the need for more aggressive evaluation and treatment of anemia. Adherence to restrictive transfusion guidelines offers an opportunity to reduce transfusion rates which may also improve short-term outcomes.

Crosstalk between ERK and MRTF-A signaling regulates TGFß1-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell-cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast-like properties with enhanced expression of the mesenchymal protein marker α-smooth muscle actin and contractile activity. Transforming growth factor (TGF)-ß1 is a well-known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal-regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFß1-induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin-related transcription factor-A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes.

Unique Mutations in the Murine Hepatitis Virus Macrodomain Differentially Attenuate Virus Replication, Indicating Multiple Roles for the Macrodomain in Coronavirus Replication.

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in nonstructural protein 3 (nsp3) that binds and hydrolyzes mono-ADP-ribose (MAR) covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutation of a highly conserved asparagine residue, which makes contact with the distal ribose of ADP-ribose. To determine if additional Mac1 activities contribute to CoV replication, we compared the replication of murine hepatitis virus (MHV) Mac1 mutants, D1329A and N1465A, to the previously mentioned asparagine mutant, N1347A. These residues contact the adenine and proximal ribose in ADP-ribose, respectively. N1465A had no effect on MHV replication or pathogenesis, while D1329A and N1347A both replicated poorly in bone marrow-derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. Interestingly, D1329A was also significantly more attenuated than N1347A in all cell lines tested. Conversely, D1329A retained some ability to block beta interferon (IFN-ß) transcript accumulation compared to N1347A, indicating that these mutations have different effects on Mac1 functions. Combining these two mutations resulted in a virus that was unrecoverable, suggesting that the combined activities of Mac1 are essential for MHV replication. We conclude that Mac1 has multiple functions that promote the replication of MHV, and that these results provide further evidence that Mac1 is a prominent target for anti-CoV therapeutics. IMPORTANCE In the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within nonstructural protein 3. It has received significant attention as a potential drug target, as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the functions of Mac1 during infection remain largely unknown. Here, using targeted mutations in different regions of Mac1, we found that Mac1 has multiple functions that promote the replication of MHV, a model CoV, and, therefore, is more important for MHV replication than previously appreciated. These results will help guide the discovery of these novel functions of Mac1 and the development of inhibitory compounds targeting this domain.

Blood products and liver transplantation: A strategy to balance optimal preparation with effective blood stewardship.

BACKGROUND: Unanticipated transfusion requirements during liver transplantation can delay lifesaving intraoperative resuscitation and strain blood bank resources. Risk-stratified preoperative blood preparation can mitigate these deleterious outcomes. STUDY DESIGN AND METHODS: A two-tiered blood preparation protocol for liver transplantation was retrospectively evaluated. Eleven binary variables served as criteria for high-risk (HR) allocation. Primary outcomes included red blood cell (RBC), plasma (FFP), and platelet (Plt) utilization. Secondary outcomes included product under- and overpreparation. Contingency tables for transfusion requirements above the population means were generated using 15 clinical variables. Modified protocols were developed and retrospectively optimized using the study population. RESULTS: Of 225 recipients, 102 received HR preoperative orders, which correlated to higher intraoperative transfusion requirements. However, univariate analysis identified only two statistical risk factors per product: Hgb ≤7.8 g/dl (p < .001) and MELD ≥38 (p = .035) for RBCs, Hgb ≤7.8 g/dl (p = .002) and acute alcoholic hepatitis (p = 0.015) for FFP, and Hgb ≤7.8 g/dl (p = .001) and normothermic liver preservation (p = .037) for Plts. Based on these findings, we developed modified protocols for individual products, which were evaluated retrospectively for their effectiveness at reducing under-preparatory events while limiting product overpreparation. Cohort statistics were used to define the preparation strategy for each protocol. Retrospective comparative analysis demonstrated the superiority of the modified protocols by improving the under-preparation rate from 24% to <10% for each product, which required a 1.56-fold and 1.44-fold increase in RBC and FFP overpreparation, respectively. Importantly, there was no difference in Plt overpreparation. DISCUSSION: We report translatable data-driven blood bank preparation protocols for liver transplantation.

Defining postoperative transfusion thresholds in liver transplant recipients: A novel retrospective approach.

BACKGROUND: The optimal transfusion threshold for most patient populations has been defined as hematocrit (HCT) <21%. However, some specific patient populations are known to benefit from higher transfusion thresholds. To date, the optimal postoperative transfusion threshold for patients undergoing liver transplant has not been determined. To define the ideal transfusion threshold for liver transplant patients, we designed a retrospective study of 496 liver transplant recipients. METHODS: Using HCT prior to discharge as a surrogate marker for transfusion thresholds we grouped patients into three groups of transfusion thresholds (HCT <21%, <24%, and >30%). Transfusion rates (intra- and postoperative), graft and patient survival, and complications requiring readmission were compared between groups. RESULTS: Ninety-two percent of patients were transfused during their hospital stay. Graft survival, patient survival, and rates of readmission within 30 days of discharge were no different between the three discharge HCT groups. Patients discharged with HCT >30% were less likely to be readmitted with infectious complications; however, this group also had the lowest model of end-stage liver (MELD) score at time of transplantation and were less likely to have received a transfusion during their hospital stay. CONCLUSION: Transfusion thresholds of HCT <24%, and potentially as low as 21% are acceptable in postoperative liver transplant recipients. The conduct of a randomized clinical trial, as supported by these data, will be necessary to support the use of lower thresholds.

Persistent viral RNA shedding after COVID-19 symptom resolution in older convalescent plasma donors.

INTRODUCTION: The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for a worldwide pandemic. While the medical community understands the mode of viral transmission, less is known about how long viral shedding occurs once viral symptoms have resolved. Our objective was to determine how long the SARS-CoV-2 remains detectable following self-reporting of viral symptom resolution. METHODS: This study was approved by the University of Wisconsin Institutional Review Board. A cohort of patients who were previously SARS-CoV-2 positive less than 28 days after self-reported symptom resolution were retested for proof of viral recovery by nasal swab reverse transcriptase polymerase chain reaction for SARS-CoV-2 RNA. RESULTS: A total of 152 potential participants were screened, of which 5 declined, 54 were ineligible, and 93 were recruited; 86 of 93 completed testing. Eleven of 86 (13%) were still positive at a median of 19 days (range, 12-24 days) after symptom resolution. Positive participants were significantly older than negative participants (mean, 54 years; 95% confidence interval [CI], 44-63 vs 42 years; 95% CI, 38-46; P = .024). CT values were significantly, inversely associated with age (ß = -.04; r2 = 0.389; P = .04). The number of days since symptom recovery was not apparently different between positive and negative participants. CONCLUSION: We found evidence of persistent viral shedding in nasopharyngeal secretions more than 2 weeks after resolution of symptoms from confirmed COVID-19 infection. Persistent shedding was more common in older participants, and viral load was higher among older positive participants. These results underscore the necessity of testing COVID-19 convalescent plasma donors less than 28 days after symptom resolution.

Electronic clinical decision support to facilitate a change in clinical practice: Small details can make or break success.

BACKGROUND: The use of electronic clinical decision support (CDS) is becoming common to change historically common clinical practices considered outdated by current guidelines. Preimplementation design of CDS tools is key to their success in changing clinical behaviors. Unfortunately, there are no established protocols for CDS tool development, and CDS failure can result from even small design flaws. This paper describes an example of a design oversight and how correction resulted in CDS success. STUDY DESIGN AND METHODS: We performed a retrospective review of compliance with a CDS tool to encourage the use of prothrombin complex concentrate over plasma transfusion for the emergent reversal of warfarin. We identified a potential design flaw, made the necessary modifications, and repeated the compliance review. RESULTS: After CDS, plasma orders declined by 150 units/mo; however, 48% of orders placed for non-warfarin coagulopathy were still for warfarin reversal. Hospital-wide, this noncompliance was 36% and was 80% in the emergency department. By simply relocating the qualifier "NOT on warfarin" from the end to the beginning of the order, noncompliance for warfarin reversal was reduced to 5% (P < .0001 by chi-square). CONCLUSIONS: The successful use of electronic clinical decision support in the electronic medical record can depend on optimal design. Missing even small design elements such as the positioning of key terms within the tool can result in an ineffective CDS. Important design strategies to avoid poor performance are discussed as they relate to the CDS tool we describe.

Transition-Metal Catalysis of Triene 6π Electrocyclization: The π-Complexation Strategy Realized.

Triene 6π electrocyclization, wherein a conjugated triene undergoes a concerted stereospecific cycloisomerization to a cyclohexadiene, is a reaction of great historical and practical significance. In order to circumvent limitations imposed by the normally harsh reaction conditions, chemists have long sought to develop catalytic variants based upon the activating power of metal-alkene coordination. Herein, we demonstrate the first successful implementation of such a strategy by utilizing [(C5 H5 )Ru(NCMe)3 ]PF6 as a precatalyst for the disrotatory 6π electrocyclization of highly substituted trienes that are resistant to thermal cyclization. Mechanistic and computational studies implicate hexahapto transition-metal coordination as responsible for lowering the energetic barrier to ring closure. This work establishes a foundation for the development of new catalysts for stereoselective electrocyclizations.

Stored platelet number and viscoelastic maximum amplitude are not altered by warming or rapid infusion.

BACKGROUND: Platelet transfusion is an important aspect of hemostatic resuscitation. Leading textbooks recommend never infusing platelets through warmers or rapid infusers, but there is no evidence to justify this position. MATERIALS AND METHODS: We obtained units of apheresis platelets in plasma from our hospital blood bank and drew a baseline sample from every unit. In the warmer arm, an aliquot from each unit was injected into a fluid warmer heated to 41°C (Ranger, 3M Corporation). After 5 minutes' incubation, the aliquot was withdrawn and sampled. In the infuser arm, we ran the remainder of the unit through a rapid infuser (RI-2, Belmont Instrument Corporation) at 500 mL/min while warmed, and obtained a sample from the outflow line. A platelet count and viscoelastic maximum amplitude (Haemonetics) was measured from every sample. RESULTS: We observed no clotting or device malfunctions. Average postwarmer temperature was 41.8°C (range, 41.0-43.0). There was no significant difference in postwarmer platelet count or viscoelastic maximum amplitude. Average postinfuser temperature was 37.4°C (range, 36.1-39.0). All units reached the goal infusion rate of 500 mL/min. There was a small increase in postinfuser platelet count. There was no significant change in postinfuser viscoelastic maximum amplitude. CONCLUSION: We were unable to detect any effect of warming or rapid infusion on the number or viscoelastic maximum amplitude of stored apheresis platelets. Contrary to common teaching, these results suggest that rapid infusion and warming does not meaningfully harm apheresis platelets.

MUC16 suppresses human and murine innate immune responses.

OBJECTIVE: MUC16, the mucin that contains the CA125 epitopes, suppresses the cytolytic responses of human NK cells and inhibits the efficacy of therapeutic antibodies. Here, we provide further evidence of the regulatory role of MUC16 on human and murine NK cells and macrophages. METHODS: Target cell cytolysis and doublet formation assays were performed to assess effects of MUC16 on human NK cells. The effect of MUC16 on ovarian tumor growth was determined in a mouse model by monitoring survival and ascites formation. Innate immune cells from spleens and peritoneal cavities of mice were isolated and stimulated in vitro with anti-CD40 antibody, lipopolysaccharide and IFN-γ and their ability to cytolyse MUC16 expressing and non-expressing cells was determined. RESULTS: We confirm that MUC16 inhibits cytolysis by human NK cells as well as the formation of NK-tumor conjugates. Mice implanted with MUC16-knockdown OVCAR-3 show >2-fold increase in survival compared to controls. Murine NK cells and macrophages are more efficient at lysing MUC16-knockdown cells. In vitro cytotoxicity assays with NK cells and macrophages isolated from mice stimulated with anti-CD40 antibody showed 2-3-fold increased activity against the MUC16-knockdown cells as compared to matching target cells expressing this mucin. Finally, knockdown of MUC16 increased the susceptibility of cancer cells to ADCC by murine splenocytes. CONCLUSIONS: For the first time, we demonstrate the immunoregulatory effects of MUC16 on murine NK cells and macrophages. Our study implies that the immunoregulatory role of MUC16 on murine NK cells and macrophages should be considered when examining the biology of MUC16 in mouse models.

Acid-Induced Liberation of Polysubstituted Cyclopentadiene Ligands from Cyclopentadienyl Cobalt: A [2 + 2 + 1] Cycloaddition Route toward 1,2,4-Trisubstituted Cyclopentadienes.

Here, we report that trifluoroacetic acid (TFAH) induces demetallation and protodesilylation of the cyclopentadiene ligand in cobalt-η4-cyclopentadiene complexes of general formula [(η5-C5H5)Co(η4-exo-C(TMS)═C(SO2Ph)CH═CRCH(CO2Et))] (1-Ph, R = Ph; 1-ArtBu, R = p-C6H4tBu; 1-ArNMe2, R = p-C6H4NMe2; and 1-Me, R = Me). The trisubstituted cyclopentadiene products are isolated as a mixture of two tautomers, [(CH2C(SO2Ph)═CHC(CO2Et)═CR)] (8-R-A) and [(CH═C(SO2Ph)CH2C(CO2Et)═CR)] (8-R-B). The endo isomer, [(η5-C5H5)Co(η4-endo-C(TMS)═C(SO2Ph)CH═CPhCH(CO2Et))] (1-Ph-endo), also undergoes demetallation and protodesilylation to give 8-Ph-A and 8-Ph-B in excellent yield. The cobalt-cyclopentadiene complex, [(η5-C5H5)Co(η4-exo-C(TMS)═C(SO2Ph)CH═C(CO2Me)CH(CO2Et))] (1-CO2Me), undergoes demetallation and protodesilylation upon treatment with TFAH to give a hydrogen-bonded fulvenol (8-CO2Me). Liberation of the ethoxy-substituted cyclopentadiene ligand of [(η5-C5H5)Co(η4-exo-C(TMS)═C(SO2Ph)CH═C(OEt)CH(CO2Et))] (1-OEt) leads to formation of a cyclopentenone derivative (11). Thermolysis of 8-Ph-A/8-Ph-B in the presence of maleimide leads to a highly functionalized Diels-Alder adduct, whereas 8-Ph-A/8-Ph-B serves as precursors to trisubstituted ruthenocenes by in situ deprotonation and reaction with [(η5-C5R5)Ru(NCMe)3]PF6 (16-H, R = H; 16-Me, R = Me).

Impact of the hematocrit value of the blood prime unit on patient hematocrit values after therapeutic plasma exchange in children weighing 10 kg or less.

We report the impact of measuring the hematocrit (HCT) of blood prime units (BPUs) on postprocedure patient HCT values in a small child with transverse myelitis undergoing therapeutic plasma exchange (TPE). Initially, the BPU HCT values were not measured, according to our apheresis policy of using our blood center's estimated HCT value. This approach resulted in unexpected increasing elevations of our patient's post-TPE HCT after the first two TPE procedures. Subsequent measurement of the BPU HCT prior to use stabilized the patient's post-TPE HCT. To our knowledge, this is the first case report describing the impact of using the measured BPU HCT vs the estimated HCT for very small children undergoing therapeutic apheresis. Our standard operating procedure for very small children has been updated after this patient's case to include measurements of the HCT values of BPUs for children who weigh 10 kg or less.

Neoadjuvant chemotherapy is associated with a high rate of perioperative blood transfusion at the time of interval cytoreductive surgery.

BACKGROUND: The oncologic safety of allogeneic blood transfusion in ovarian cancer patients is unknow. We sought to determine the prevalence and oncologic safety of perioperative allogeneic blood transfusion during interval cytoreduction surgery among women receiving neoadjuvant chemotherapy for ovarian cancer. METHODS: We utilized retrospective chart review to identify a cohort of patients undergoing interval cytoreduction at a large academic tertiary referral center. We compared outcomes in patients who were exposed to perioperative blood transfusion compared with patients who were not exposed. Our primary endpoint was progression free survival; our secondary endpoint was overall survival. Baseline clinical characteristics were collected for patients in each group. RESULTS: Sixty-six women were included in the final cohort of women undergoing interval cytoreductive surgery after NACT. A total of 51 women (77%) were exposed to allogeneic perioperative pRBC transfusion. Fifteen women (23%) were not exposed to transfusion. The baseline characteristics were generally well matched. Women who were not exposed to a perioperative blood transfusion were more likely to have a normalized CA125 prior to undergoing cytoreductive surgery. Preoperative hemoglobin concentration was lower in the transfusion group (10.5 g/dLvs 11.5 g/dL, p < 0.009). Perioperative transfusion was not associated with a significant difference in progression free survival (PFS = 7.6 months for transfused, 9.4 months for not transfused; log-rank test p = 0.4617). Similarly, there was no observed difference between groups for overall survival (OS = 23.6 months for transfused, 22.5 months for not transfused; log-rank test p = 0.1723). CONCLUSIONS: Women undergoing neoadjuvant chemotherapy for ovarian cancer are at high risk of exposure to blood transfusion at the time of interval cytoreductive surgery. Future studies will continue to evaluate the safety and impact of transfusion on ovarian cancer survival in this at risk population.

Outcomes of red blood cell transfusions prescribed in organ donors by the Digital Intern, an electronic decision support algorithm.

BACKGROUND: The Digital Intern (DI) is an electronic decision support tool for the management of organ donors. One algorithm determines the dose, in units of red blood cells to be transfused, based on hematocrit (Hct) thresholds and targets. The effectiveness of the transfusion dose calculated by the DI in terms of achieving the selected Hct target and the duration of the targeted dose is not known. STUDY DESIGN AND METHODS: This was a retrospective study to describe the outcomes of transfusions prescribed by the DI. Pre- and posttransfusion Hct levels were compared to define response and all posttransfusion Hct values were plotted to evaluate the duration of the prescribed dose. RESULTS: A total of 120 organ donors were studied and 22 donors had 28 transfusions (six were transfused twice). The transfused donors were a mix of trauma and medical admissions and brain death and cardiac death donors. The transfusion target of 24% Hct was attained in 96% of transfusions. The mean number of units transfused was 1.4 and the mean time from transfusion to procurement was 19.8 hours. There was a decline in Hct over time after transfusion in all but one case with a mean decline of 1.9% Hct over 13 hours. Six donors were transfused twice, likely due to a longer donor time period (41.7 hr vs. 27 hr). CONCLUSIONS: The DI provided transfusion dosing that achieved the desired threshold in the majority of organ donors transfused. Ongoing work focuses on application of this technology to transfusions in general patient populations.

The blood utilization calculator, a target-based electronic decision support algorithm, increases the use of single-unit transfusions in a large academic medical center.

BACKGROUND: Electronic decision support has been used to reduce use of red blood cell (RBC) transfusion. With the goal of reducing transfusions, we modified our RBC orders to default to 1 unit. Next, we created a target-based algorithm, the blood utilization calculator or BUC, to calculate a dose in units, based on initial hemoglobin or hematocrit and weight. STUDY DESIGN AND METHODS: RBC orders defaulted to 1 unit in March 2016 and the BUC was implemented in July 2016. This gave three periods to compare old orders (before intervention), new orders (1-unit default), and the BUC period. A hospital dashboard that tracks blood product orders was queried to determine changes in single-unit transfusions between periods. Changes in transfusions were compared by analysis of variance. Acceptance of the BUC dosage recommendation was studied in both medical-based and surgical-based specialties. RESULTS: The number of single-unit transfusions showed significant increases after each of the two interventions studied from 247 ± 19 before interventions to 358 ± 19 and then to 445 ± 141-unit transfusions/month (p < 0.0001). The ratio of 1-unit to 2-unit transfusions increased from 0.72 to 1.67 (p < 0.0001) and we observed a 19% overall reduction in units transfused. The BUC recommendation was accepted in 49% of orders. CONCLUSIONS: One-unit default orders and implementation of the BUC resulted in a significant increase in the use of single-unit transfusions. Improvement in the rate of acceptance of the BUC recommendation should further increase the use of single-unit transfusions.

Peri-operative allogeneic blood transfusion is associated with poor overall survival in advanced epithelial ovarian Cancer; potential impact of patient blood management on Cancer outcomes.

BACKGROUND: Transfusion related immune modulation associated with red blood cell (RBC) transfusion is thought to result in decreased cancer survival. Results in epithelial ovarian cancer (EOC) have been mixed however most suggest worse oncologic outcomes in patients who were transfused at the time of debulking surgery. The impact of restrictive transfusion strategies on this patient population is currently not known. METHODS: We conducted a retrospective study of women with EOC. The study population was divided into two groups based on whether they were transfused RBCs during the peri-operative period or not. Clinical characteristics and prognosticators were compared between groups. Overall survival was compared between groups based on transfusion status and other known prognostic factors. Cox proportional hazard modeling was used to examine the association between the prognostic factors and the study endpoint. RESULTS: Sixty-six percent of women were transfused. Transfusion was associated with CA125, the use of neoadjuvant chemotherapy (NACT), surgical blood loss, and anemia. The mean pre-transfusion Hgb was 7.8 + 0.6 g/dL and 94% had a hemoglobin level greater than the transfusion threshold of 7 g/dL. RBC transfusion, suboptimal debulking, anemia, and NACT were associated with decreased survival. Only RBC transfusion and suboptimal debulking status remained significant in a multivariate model. CONCLUSIONS: Peri-operative RBC transfusion compromises survival in ovarian cancer supporting the need to minimize the use of transfusion at the time of debulking surgery. Adherence to evidence-based transfusion guidelines offers an opportunity to reduce transfusion rates in this population with a resulting positive influence on survival.

Neoadjuvant chemotherapy is associated with more anemia and perioperative blood transfusions than primary debulking surgery in women with advanced stage ovarian cancer.

OBJECTIVE: The purpose of this case-controlled study was to determine the prevalence of anemia and incidence of perioperative blood transfusions in patients undergoing treatment for advanced ovarian cancer with neoadjuvant chemotherapy (NACT) or primary debulking surgery (PDS). METHODS: We performed a single institution review of patients diagnosed with stage IIIB-IVB epithelial ovarian cancer between 2010 and 2013 undergoing either NACT or PDS. Anemia was defined as a hemoglobin (Hgb) concentration of ≤11.5 g/dL. Continuous variables were compared by student t-test and binary variables compared via chi square analysis. RESULTS: One hundred thirty-one women were included, 66 treated with NACT and 65 treated with PDS. Average Hgb prior to surgery was lower in women who received NACT (10.7 g/dL vs 12.8 g/dL, p < 0.0001). Women treated with NACT had a decrease in mean Hgb during chemotherapy treatment (11.8 g/dL at diagnosis to 10.7 g/dL preoperatively). Seventy-seven percent of NACT patients were anemic prior to surgery compared to 15% of patients prior to PDS (p < 0.001). Mean EBL at debulking was higher in patients selected for PDS (871 mL) than NACT (544 mL); however, the perioperative transfusion rate was higher during interval debulking surgeries (NACT 77% vs PDS 56%, p = 0.01). CONCLUSION: Women selected for NACT were more likely to be anemic at diagnosis and became progressively anemic during NACT. Despite less blood loss during debulking surgery, NACT patients receive more blood transfusions perioperatively than patients undergoing PDS. This represents a potential opportunity for therapeutic intervention during NACT to correct anemia prior to interval debulking surgery.