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.

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.

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.

Standardization of transfusion practice in organ donors using the Digital Intern, an electronic decision support algorithm.

BACKGROUND: Prospective clinical trials support restrictive thresholds for red blood cell (RBC) transfusion. Nonsurvivable donors are a major source of organs for transplantation. The Digital Intern (DI) is a computer algorithm to standardize donor care that includes a more restrictive transfusion threshold. The impact of standardized and restrictive RBC transfusion in organ donors, as determined by the DI, has not been reported. STUDY DESIGN AND METHODS: We conducted a retrospective cohort study to compare the transfusion practice of the DI (n = 100) to a historic group of physician-managed donors (n = 90). Transfusion rates, the number of units transfused, and pretransfusion laboratory values were compared between groups. The variability of these parameters was also compared between groups. Finally, the number of transplanted organs per donor in each group was compared. RESULTS: The mean time as a donor was 25.9 ± 15.2 hours and was not different between the groups. In the DI group 19% were transfused compared to 26% in the control group (p = 0.3). The number of units transfused was less in the DI group (1 unit vs. 2 units per transfusion, p = 0.03) and the pretransfusion hematocrit was lower in the DI group (23% vs. 27%, p = 0.01). The variability in the latter two parameters was significantly lower in the DI group. The number of transplanted organs per donor was similar in both groups (3.24 [DI] vs. 3.03 [control], p = 0.37). CONCLUSION: The DI provides a more standardization transfusion practice in organ donors and reduces blood use without compromising transplantable organs.

A phase 1b study of humanized KS-interleukin-2 (huKS-IL2) immunocytokine with cyclophosphamide in patients with EpCAM-positive advanced solid tumors.

BACKGROUND: Humanized KS-interleukin-2 (huKS-IL2), an immunocytokine with specificity for epithelial cell adhesion molecule (EpCAM), has demonstrated favorable tolerability and immunologic activity as a single agent. METHODS: Phase 1b study in patients with EpCAM-positive advanced solid tumors to determine the maximum tolerated dose (MTD) and safety profile of huKS-IL2 in combination with low-dose cyclophosphamide. Treatment consisted of cyclophosphamide (300 mg/m2 on day 1), and escalating doses of huKS-IL2 (0.5-4.0 mg/m2 IV continuous infusion over 4 hours) on days 2, 3, and 4 of each 21-day cycle. Safety, pharmacokinetic profile, immunogenicity, anti-tumor and biologic activity were evaluated. RESULTS: Twenty-seven patients were treated for up to 6 cycles; 26 were evaluable for response. The MTD of huKS-IL2 in combination with 300 mg/m2 cyclophosphamide was 3.0 mg/m2. At higher doses, myelosuppression was dose-limiting. Transient lymphopenia was the most common grade 3/4 adverse event (AE). Other significant AEs included hypotension, hypophosphatemia, and increase in serum creatinine. All patients recovered from these AEs. The huKS-IL2 exposure was dose-dependent, but not dose-proportional, accumulation was negligible, and elimination half-life and systemic clearance were independent of dose and time. Most patients had a transient immune response to huKS-IL2. Immunologic activity was observed at all doses. Ten patients (38%) had stable disease as best response, lasting for ≥ 4 cycles in 3 patients. CONCLUSION: The combination of huKS-IL2 with low-dose cyclophosphamide was well tolerated. Although no objective responses were observed, the combination showed evidence of immunologic activity and 3 patients showed stable disease for ≥ 4 cycles.

Uterine leiomyosarcoma diffusely express disialoganglioside GD2 and bind the therapeutic immunocytokine 14.18-IL2: implications for immunotherapy.

Uterine leiomyosarcoma comprises <1 % of uterine malignancies and is known for its clinically aggressive course. Extrapelvic recurrences are common and often lethal. No adjuvant therapies have been shown to significantly improve overall survival, highlighting the need for new and novel therapies. Our objective was to determine whether GD2-specific immunocytokine therapy may be explored for the treatment for uterine leiomyosarcoma. To do so, frozen tissue sections were obtained from the Gynecologic Oncology Group tumor bank and evaluated by immunohistochemistry (IHC) for GD2 expression using both the parent mouse monoclonal antibody 14G2A and immunocytokine 14.18-IL2 generated from the 14G2A sequence. Immunoreactivity was detected by avidin-biotin complex with DAB substrate. Specimens were reviewed by a pathologist with light microscopy and classified as negative, 1+, 2+ or 3+, compared to human melanoma cells as positive control and tissue incubated in the absence of primary antibody as negative control. GD2 was diffusely present in all evaluable samples. 10 tumors (67 %) demonstrated 3+ IHC intensity for GD2, two tumors (13 %) demonstrated 2+ intensity, and 3 (20 %) tumors demonstrated 1+ intensity. Eleven cases had sufficient tissue to assess 14.18-IL2 binding. All 11 cases bound 14.18-IL2 in a pattern identical to the parent antibody. Uterine leiomyosarcoma diffusely express GD2 and bind the therapeutic immunocytokine 14.18-IL2. This warrants further exploration to determine whether immunocytokine therapy may have a clinical role in the management of these aggressive tumors.

DcR3 binds to ovarian cancer via heparan sulfate proteoglycans and modulates tumor cells response to platinum with corresponding alteration in the expression of BRCA1.

BACKGROUND: Overcoming platinum resistance is a major obstacle in the treatment of Epithelial Ovarian Cancer (EOC). In our previous work Decoy Receptor 3 (DcR3) was found to be related to platinum resistance. The major objective of this work was to define the cellular interaction of DcR3 with EOC and to explore its effects on platinum responsiveness. METHODS: We studied cell lines and primary cultures for the expression of and the cells ability to bind DcR3. Cells were cultured with DcR3 and then exposed to platinum. Cell viability was determined by MTT assay. Finally, the cells molecular response to DcR3 was studied using real time RT-PCR based differential expression arrays, standard RT-PCR, and Western blot. RESULTS: High DcR3 in the peritoneal cavity of women with EOC is associated with significantly shorter time to first recurrence after platinum based therapy (p = 0.02). None-malignant cells contribute DcR3 in the peritoneal cavity. The cell lines studied do not secrete DcR3; however they all bind exogenous DcR3 to their surface implying that they can be effected by DcR3 from other sources. DcR3s protein binding partners are minimally expressed or negative, however, all cells expressed the DcR3 binding Heparan Sulfate Proteoglycans (HSPGs) Syndecans-2, and CD44v3. DcR3 binding was inhibited by heparin and heparinase. After DcR3 exposure both SKOV-3 and OVCAR-3 became more resistant to platinum with 15% more cells surviving at high doses. On the contrary CaOV3 became more sensitive to platinum with 20-25% more cell death. PCR array analysis showed increase expression of BRCA1 mRNA in SKOV-3 and OVCAR-3 and decreased BRCA1 expression in CaOV-3 after exposure to DcR3. This was confirmed by gene specific real time PCR and Western blot analysis. CONCLUSIONS: Non-malignant cells contribute to the high levels of DcR3 in ovarian cancer. DcR3 binds readily to EOC cells via HSPGs and alter their responsiveness to platinum chemotherapy. The paradoxical responses seen were related to the expression pattern of HSPGs available on the cells surface to interact with. Although the mechanism behind this is not completely known alterations in DNA repair pathways including the expression of BRCA1 appear to be involved.

Quality of life in cervical cancer survivors: patient and provider perspectives on common complications of cervical cancer and treatment.

OBJECTIVE: This study's objective was to quantify the impact (utility) of common complications of early cervical cancer treatment on quality of life (QOL). Utilities assigned by survivors were compared to those assigned by providers. METHODS: 30 survivors of early cervical cancer identified from our Tumor Registry and 10 gynecologic oncology providers were interviewed. Participants evaluated complications (health states) using the standard gamble (SG) and visual analogue scale (VAS). Each participant was randomly assigned to rate 5 of 13 health states. Mixed-effects linear models were used to generate confidence intervals for utility means, and evaluate the effect of group (survivors versus providers). Higher utilities indicate the health state is closer to perfect health. RESULTS: Survivors and providers mean ages were similar (44 and 40). Mean time from diagnosis was 6.7 years. 28 of 30 survivors had no evidence of disease. 56% of survivors had complications. Using SG, providers consistently assigned utilities 7% higher than survivors (p=0.035) for all health states except "ileostomy", which survivors rated higher than providers. Survivors assigned the lowest utility to small bowel obstruction (SBO) (fixable without an ostomy) and ureteral obstruction (UO). Survivors rated SBO 16% and UO 21% lower than providers. Personal history of complications or higher stage did not have a consistent effect on QOL adjustments. DISCUSSION: Providers assign higher utilities than survivors to health states. Providers and survivors diverge on which complications impact QOL the most. Data on patient preferences should be considered when weighing treatment options with similar survival but different associated complications.

Intraoperative autologous blood transfusion use during radical hysterectomy for cervical cancer: long-term follow-up of a prospective trial.

PURPOSE: A primary operative complication of radical hysterectomy for cervical cancer is hemorrhage. Intraoperative autologous blood transfusion (ABT) may be beneficial in reducing the need for homologous blood transfusion. METHODS: Our institution published a prospective cohort study examining the use of ABT in cervical cancer patients undergoing radical hysterectomy in 1995. Patients who were initially consented to participate in this prospective trial using intraoperative ABT (cell saver) were evaluated with a median follow-up of 3 years. We sought to update this original report with 16-year follow-up data collected from the clinical charts, Tumor Registry, and the Social Security Death Index. RESULTS: Two groups of patients undergoing radical hysterectomy were compared: patients who received ABT, and those who did not. Of the 71 original patients, all were included in this updated review, with an average follow-up of 12.4 years for both groups. Originally, thirty-one patients received an ABT. In this group, 1 patient was lost to follow-up, and 4 (12.9 %) are deceased including 1 (3 %) with disease. In the non-autologous group, there were 7 (17.5 %) patient deaths, including 3 (7.5 %) with disease. Eighty-three percent were alive after 12 years in both groups. The ABT group had 1 patient (3 %) who developed a secondary malignancy, a colon adenocarcinoma. The non-autologous group had 2 patients (5 %) who developed a secondary malignancy; one patient developed multiple myeloma and one patient developed a verrucous cancer of the tongue. CONCLUSIONS: Autologous blood transfusion during radical hysterectomy for cervical cancer appears safe and effective.

Anti-SARS-CoV-2 IgG and IgA antibodies in COVID-19 convalescent plasma do not enhance viral infection.

The novel coronavirus, SARS-CoV-2 that causes COVID-19 has resulted in the death of nearly 4 million people within the last 18 months. While preventive vaccination, and monoclonal antibody therapies have been rapidly developed and deployed, early in the pandemic the use of COVID-19 convalescent plasma (CCP) was a common means of passive immunization with a theoretical risk of antibody-dependent enhancement (ADE) of viral infection. Though vaccines elicit a strong and protective immune response and transfusion of CCP with high titers of neutralization activity are correlated with better clinical outcomes, the question of whether antibodies in CCP can enhance infection of SARS-CoV-2 has not been directly addressed. In this study, we analyzed for and observed passive transfer of neutralization activity with CCP transfusion. Furthermore, to specifically understand if antibodies against the spike protein (S) enhance infection, we measured the anti-S IgG, IgA, and IgM responses and adapted retroviral-pseudotypes to measure virus neutralization with target cells expressing the ACE2 virus receptor and the Fc alpha receptor (FcαR) or Fc gamma receptor IIA (FcγRIIA). Whereas neutralizing activity of CCP correlated best with higher titers of anti-S IgG antibodies, the neutralizing titer was not affected when Fc receptors were present on target cells. These observations support the absence of antibody-dependent enhancement of infection (ADE) by IgG and IgA isotypes found in CCP. The results presented, therefore, not only supports the therapeutic use of currently available antibody-based treatment, including the continuation of CCP transfusion strategies, but also the use of various vaccine platforms in a prophylactic approach.

Predicting the Efficacy of COVID-19 Convalescent Plasma Donor Units with the Lumit Dx anti-Receptor Binding Domain Assay.

BACKGROUND: The novel coronavirus SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.5 million people, but no cure exists. Although passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable therapeutic option, the selection of optimal units for therapy in a timely fashion remains a barrier. STUDY DESIGN AND METHODS: Since virus neutralization is a necessary characteristic of plasma that can benefit recipients, the neutralizing titers of plasma samples were measured using a retroviral-pseudotype assay. Binding antibody titers to the spike (S) protein were also determined by a clinically available serological assay (Ortho-Vitros total IG), and an in-house ELISA. The results of these assays were compared to a measurement of antibodies directed to the receptor binding domain (RBD) of the SARS-CoV2 S protein (Promega Lumit Dx). RESULTS: All measures of antibodies were highly variable, but correlated, to different degrees, with each other. However, the anti-RBD antibodies correlated with viral neutralizing titers to a greater extent than the other antibody assays. DISCUSSION: Our observations support the use of an anti-RBD assay such as the Lumit Dx assay, as an optimal predictor of the neutralization capability of CCP.

Anti-SARS-CoV-2 IgG and IgA antibodies in COVID-19 convalescent plasma do not facilitate antibody-dependent enhance of viral infection.

The novel coronavirus SARS-CoV2, which causes COVID-19, has resulted in the death of nearly 4 million people within the last 18 months. While preventive vaccination and monoclonal antibody therapies have been rapidly developed and deployed, early in the pandemic the use of COVID-19 convalescent plasma (CCP) was a common means of passive immunization, with the theoretical risk of antibody-dependent enhancement (ADE) of viral infection remaining undetermined. Though vaccines elicit a strong and protective immune response, and transfusion of CCP with high titers of neutralization activity are correlated with better clinical outcomes, the question of whether antibodies in CCP can enhance infection of SARS-CoV2 has not been directly addressed. In this study, we analyzed for and observed passive transfer of neutralization activity with CCP transfusion. Furthermore, to specifically understand if antibodies against the spike protein (S) enhance infection, we measured the anti-S IgG, IgA, and IgM responses and adapted retroviral-pseudotypes to measure virus neutralization with target cells expressing the ACE2 virus receptor and the Fc alpha receptor (FcαR) or Fc gamma receptor IIA (FcγRIIA). Whereas neutralizing activity of CCP correlated best with higher titers of anti-S IgG antibodies, the neutralizing titer was not affected when Fc receptors were present on target cells. These observations support the absence of antibody-dependent enhancement of infection (ADE) by IgG and IgA isotypes found in CCP. The results presented, therefore, support the clinical use of currently available antibody-based treatment including the continued study of CCP transfusion strategies.

Predicting the efficacy of COVID-19 convalescent plasma donor units with the Lumit Dx anti-receptor binding domain assay.

BACKGROUND: The novel coronavirus SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.5 million people, but no cure exists. Although passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable therapeutic option, the selection of optimal units for therapy in a timely fashion remains a barrier. STUDY DESIGN AND METHODS: Since virus neutralization is a necessary characteristic of plasma that can benefit recipients, the neutralizing titers of plasma samples were measured using a retroviral-pseudotype assay. Binding antibody titers to the spike (S) protein were also determined by a clinically available serological assay (Ortho-Vitros total IG), and an in-house ELISA. The results of these assays were compared to a measurement of antibodies directed to the receptor binding domain (RBD) of the SARS-CoV2 S protein (Promega Lumit Dx). RESULTS: All measures of antibodies were highly variable, but correlated, to different degrees, with each other. However, the anti-RBD antibodies correlated with viral neutralizing titers to a greater extent than the other antibody assays. DISCUSSION: Our observations support the use of an anti-RBD assay such as the Lumit Dx assay, as an optimal predictor of the neutralization capability of CCP.

Donor Anti-Spike Immunity is Related to Recipient Recovery and Can Predict the Efficacy of Convalescent Plasma Units.

BACKGROUND: The novel coronavirus, SARS-CoV2 that causes COVID-19 has resulted in the death of more than 2.31 million people within the last year and yet no cure exists. Whereas passive immunization with COVID-19 convalescent plasma (CCP) provides a safe and viable option, selection of optimal units for therapy and lack of clear therapeutic benefit from transfusion remain as barriers to the use of CCP. STUDY DESIGN AND METHODS: To identify plasma that is expected to benefit recipients, we measured anti-SARS-CoV2 antibody levels using clinically available serological assays and correlated with the neutralizing activity of CCP from donors. Neutralizing titer of plasma samples was measured by assaying infectivity of SARS-CoV-2 spike protein pseudotyped retrovirus particles in the presence of dilutions of plasma samples. We also used this assay to identify evidence of passive transfusion of neutralizing activity in CCP recipients. RESULTS: Viral neutralization and anti-spike protein antibodies in 109 samples from 87 plasma donors were highly varied but modestly correlated with each other. Recipients who died of COVID-19 were found to have been transfused with units with lower anti-spike antibody levels and neutralizing activity. Passive transfer of neutralization activity was documented in 62% of antibody naive plasma recipients. CONCLUSIONS: Since viral neutralization is the goal of CCP transfusion, our observations not only support the use of anti-spike SARS-CoV2 serology tests to identify beneficial CCP units, but also support the therapeutic value of convalescent plasma with high titers of anti-spike antibodies.

MUC16 provides immune protection by inhibiting synapse formation between NK and ovarian tumor cells.

BACKGROUND: Cancer cells utilize a variety of mechanisms to evade immune detection and attack. Effective immune detection largely relies on the formation of an immune synapse which requires close contact between immune cells and their targets. Here, we show that MUC16, a heavily glycosylated 3-5 million Da mucin expressed on the surface of ovarian tumor cells, inhibits the formation of immune synapses between NK cells and ovarian tumor targets. Our results indicate that MUC16-mediated inhibition of immune synapse formation is an effective mechanism employed by ovarian tumors to evade immune recognition. RESULTS: Expression of low levels of MUC16 strongly correlated with an increased number of conjugates and activating immune synapses between ovarian tumor cells and primary naïve NK cells. MUC16-knockdown ovarian tumor cells were more susceptible to lysis by primary NK cells than MUC16 expressing controls. This increased lysis was not due to differences in the expression levels of the ligands for the activating receptors DNAM-1 and NKG2D. The NK cell leukemia cell line (NKL), which does not express KIRs but are positive for DNAM-1 and NKG2D, also conjugated and lysed MUC16-knockdown cells more efficiently than MUC16 expressing controls. Tumor cells that survived the NKL challenge expressed higher levels of MUC16 indicating selective lysis of MUC16(low) targets. The higher csMUC16 levels on the NKL resistant tumor cells correlated with more protection from lysis as compared to target cells that were never exposed to the effectors. CONCLUSION: MUC16, a carrier of the tumor marker CA125, has previously been shown to facilitate ovarian tumor metastasis and inhibits NK cell mediated lysis of tumor targets. Our data now demonstrates that MUC16 expressing ovarian cancer cells are protected from recognition by NK cells. The immune protection provided by MUC16 may lead to selective survival of ovarian cancer cells that are more efficient in metastasizing within the peritoneal cavity and also at overcoming anti-tumor innate immune responses.