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BACKGROUNDCOVID-19 convalescent plasma (CCP) virus-specific antibody levels that translate into recipient posttransfusion antibody levels sufficient to prevent disease progression are not defined.METHODSThis secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double-blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low posttransfusion antibody levels was established by 2 methods: (i) analyzing virus neutralization-equivalent anti-Spike receptor-binding domain immunoglobulin G (anti-S-RBD IgG) responses in donors or (ii) receiver operating characteristic (ROC) curve analysis.RESULTSSARS-CoV-2 anti-S-RBD IgG antibody was volume diluted 21.3-fold into posttransfusion seronegative recipients from matched donor units. Virus-specific antibody delivered was approximately 1.2 mg. The high-antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP-recipient analysis for antibody thresholds correlated to reduced hospitalizations found a statistical significant association between early transfusion and high antibodies versus all other CCP recipients (or control plasma), with antibody cutoffs established by both methods-donor-based virus neutralization cutoffs in posttransfusion recipients (0/85 [0%] versus 15/276 [5.6%]; P = 0.03) or ROC-based cutoff (0/94 [0%] versus 15/267 [5.4%]; P = 0.01).CONCLUSIONIn unvaccinated, seronegative CCP recipients, early transfusion of plasma units in the upper 30% of study donors' antibody levels reduced outpatient hospitalizations. High antibody level plasma units, given early, should be reserved for therapeutic use.TRIAL REGISTRATIONClinicalTrials.gov NCT04373460.FUNDINGDepartment of Defense (W911QY2090012); Defense Health Agency; Bloomberg Philanthropies; the State of Maryland; NIH (3R01AI152078-01S1, U24TR001609-S3, 1K23HL151826NIH); the Mental Wellness Foundation; the Moriah Fund; Octapharma; the Healthnetwork Foundation; the Shear Family Foundation; the NorthShore Research Institute; and the Rice Foundation.

Importance: This study examined the role that cytokines may have played in the beneficial outcomes found when outpatient individuals infected with SARS-CoV-2 were transfused with COVID-19 convalescent plasma (CCP) early in their infection. We found that the pro-inflammatory cytokine IL-6 decreased significantly faster in patients treated early with CCP. Participants with COVID-19 treated with CCP later in the infection did not have the same effect. This decrease in IL-6 levels after early CCP treatment suggests a possible role of inflammation in COVID-19 progression. The evidence of IL-6 involvement brings insight into the possible mechanisms involved in CCP treatment mitigating SARS-CoV-2 severity.

Background: COVID-19 convalescent plasma (CCP) is an important therapeutic option for outpatients at high risk of hospitalization from SARS-CoV-2 infection. We assessed the safety of outpatient CCP transfusions administered during clinical trials., Study Design and Methods: We analyzed data pertaining to transfusion-related reactions from two randomized controlled trials in the U.S. that evaluated the efficacy of CCP versus control plasma in various ambulatory settings. Multivariable logistic regression was used to assess whether CCP was associated with transfusion reactions, after adjusting for potential confounders., Results: The combined study reported 79/1351 (5.9%) adverse events during the transfusion visit, with the majority 62/1351 (4.6%) characterized by mild, allergic-type findings of urticaria, and/or pruritus consistent with minor allergic transfusion reactions; the other reported events were attributed to the patients' underlying disease, COVID-19, or vasovagal in nature. We found no difference in the likelihood of allergic transfusion reactions between those receiving CCP versus control plasma (adjusted odds ratio [AOR], 0.75; 95% CI, 0.43-1.31). Risk of urticaria and/or pruritus increased with a pre-existing diagnosis of asthma (AOR, 2.33; 95% CI, 1.16-4.67). We did not observe any CCP-attributed antibody disease enhancement in participants with COVID-19 or increased risk of infection. There were no life-threatening severe transfusion reactions and no patients required hospitalization related to transfusion-associated complications., Discussion: Outpatient plasma administration was safely performed for nearly 1400 participants. CCP is a safe therapeutic option for outpatients at risk of hospitalization from COVID-19., (© 2023 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: Cytokines and chemokines play a critical role in the response to infection and vaccination. We aimed to assess the longitudinal association of COVID-19 vaccination with cytokine and chemokine concentrations and trajectories among people with SARS-CoV-2 infection., Methods: In this longitudinal, prospective cohort study, blood samples were used from participants enrolled in a multi-centre randomised trial assessing the efficacy of convalescent plasma therapy for ambulatory COVID-19. The trial was conducted in 23 outpatient sites in the USA. In this study, participants (aged ≥18 years) were restricted to those with COVID-19 before vaccination or with breakthrough infections who had blood samples and symptom data collected at screening (pre-transfusion), day 14, and day 90 visits. Associations between COVID-19 vaccination status and concentrations of 21 cytokines and chemokines (measured using multiplexed sandwich immunoassays) were examined using multivariate linear mixed-effects regression models, adjusted for age, sex, BMI, hypertension, diabetes, trial group, and COVID-19 waves (pre-alpha or alpha and delta)., Findings: Between June 29, 2020, and Sept 30, 2021, 882 participants recently infected with SARS-CoV-2 were enrolled, of whom 506 (57%) were female and 376 (43%) were male. 688 (78%) of 882 participants were unvaccinated, 55 (6%) were partly vaccinated, and 139 (16%) were fully vaccinated at baseline. After adjusting for confounders, geometric mean concentrations of interleukin (IL)-2RA, IL-7, IL-8, IL-15, IL-29 (interferon-λ), inducible protein-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α were significantly lower among the fully vaccinated group than in the unvaccinated group at screening. On day 90, fully vaccinated participants had approximately 20% lower geometric mean concentrations of IL-7, IL-8, and vascular endothelial growth factor-A than unvaccinated participants. Cytokine and chemokine concentrations decreased over time in the fully and partly vaccinated groups and unvaccinated group. Log 10 cytokine and chemokine concentrations decreased faster among participants in the unvaccinated group than in other groups, but their geometric mean concentrations were generally higher than fully vaccinated participants at 90 days. Days since full vaccination and type of vaccine received were not correlated with cytokine and chemokine concentrations., Interpretation: Initially and during recovery from symptomatic COVID-19, fully vaccinated participants had lower concentrations of inflammatory markers than unvaccinated participants suggesting vaccination is associated with short-term and long-term reduction in inflammation, which could in part explain the reduced disease severity and mortality in vaccinated individuals., Funding: US Department of Defense, National Institutes of Health, Bloomberg Philanthropies, State of Maryland, Mental Wellness Foundation, Moriah Fund, Octapharma, HealthNetwork Foundation, and the Shear Family Foundation., Competing Interests: Declaration of interests KAG reports consultancy work for the Aspen Institute, Teach for America, serving as a non-paid member of a scientific advisory board for Pfizer, and writing COVID-19 management guidelines for UpToDate. AGA reports consultancy work for Implementation Group, Hirslanden Klinik, and Elsevier. ERC reports receiving unrestricted research grants from Gilead and Merck paid to the Regents of the University of California and participating in an advisory board to Theratechnologies for an unrelated topic. JSC reports consultancy work for Merck and Company in 2021. TJG reports employment by Fenwal, a Fresenius Kabi Company. LLH reports research funding to Johns Hopkins Center of American Indian Health from AstraZeneca, US Centers for Disease Control and Prevention, Merck, NIH, and Pfizer. MAH reports contracts from Gilead Sciences, Insmed, and AN2 Therapeutics to the University of Cincinnati. GSM reports research grant support from Teva, Alk-Abello, Genentech, Novartis, GlaxoSmithKline, and Sanofi-Regeneron, serving as an immediate past president of the American Academy of Allergy Asthma and Immunology, and is co-chair of the Continuous Assessment Program Examination for the American Board of Allergy and Immunology. BP reports participating in part of the COVID-19 trials and pulmonary arterial hypertension trials. JHP reports research funding from MindRhythm. JSR is a consultant and advisor with Sanofi Genzyme, and a board of directors member with the American Society for Apheresis. SK reports helping to produce educational materials related to HIV with Integritas Communications and Vindico Medical Education. AC reports serving on the scientific advisory board of SAB Biotherapeutics. EMB reports personal fees and non-financial support from Terumo BCT, Abbott Laboratories, Tegus, and UptoDate, is a member of the US Food and Drug Administration Blood Products Advisory Committee, and served on a convalescent plasma guideline panel. DH reports personal fees from Neurelis, Neurotrope, and medicolegal consulting. DJS is a founder and board member with stock options (macrolide for malaria) for AliquantumRx and reports consulting for Hemex Health and royalties for malaria diagnostic test control standards to Alere. SLH reports serving on the data monitoring committee for Pfizer. All other authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Description: Coronavirus disease 2019 convalescent plasma (CCP) has emerged as a potential treatment of COVID-19. However, meta-analysis data and recommendations are limited. The Association for the Advancement of Blood and Biotherapies (AABB) developed clinical practice guidelines for the appropriate use of CCP., Methods: These guidelines are based on 2 living systematic reviews of randomized controlled trials (RCTs) evaluating CCP from 1 January 2019 to 26 January 2022. There were 33 RCTs assessing 21 916 participants. The results were summarized using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) method. An expert panel reviewed the data using the GRADE framework to formulate recommendations., Recommendation 1 (outpatient): The AABB suggests CCP transfusion in addition to the usual standard of care for outpatients with COVID-19 who are at high risk for disease progression (weak recommendation, moderate-certainty evidence)., Recommendation 2 (inpatient): The AABB recommends against CCP transfusion for unselected hospitalized persons with moderate or severe disease (strong recommendation, high-certainty evidence). This recommendation does not apply to immunosuppressed patients or those who lack antibodies against SARS-CoV-2., Recommendation 3 (inpatient): The AABB suggests CCP transfusion in addition to the usual standard of care for hospitalized patients with COVID-19 who do not have SARS-CoV-2 antibodies detected at admission (weak recommendation, low-certainty evidence)., Recommendation 4 (inpatient): The AABB suggests CCP transfusion in addition to the usual standard of care for hospitalized patients with COVID-19 and preexisting immunosuppression (weak recommendation, low-certainty evidence)., Recommendation 5 (prophylaxis): The AABB suggests against prophylactic CCP transfusion for uninfected persons with close contact exposure to a person with COVID-19 (weak recommendation, low-certainty evidence)., Good Clinical Practice Statement: CCP is most effective when transfused with high neutralizing titers to infected patients early after symptom onset.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants severely limits available effective monoclonal antibody therapies. Effective drugs are also supply limited. COVID-19 convalescent plasma (CCP) qualified for high antibody levels effectively reduces immunocompetent outpatient hospitalization. The Food and Drug Administration currently allows outpatient CCP for the immunosuppressed. Viral-specific antibody levels in CCP can range 10- to 100-fold between donors, unlike the uniform viral-specific monoclonal antibody dosing. Limited data are available on the efficacy of polyclonal CCP to neutralize variants. We examined 108 pre-δ/pre-ο donor units obtained before March 2021, 20 post-δ COVID-19/postvaccination units, and 1 pre-δ/pre-ο hyperimmunoglobulin preparation for variant-specific virus (vaccine-related isolate [WA-1], δ, and ο) neutralization correlated to Euroimmun S1 immunoglobulin G antibody levels. We observed a two- to fourfold and 20- to 40-fold drop in virus neutralization from SARS-CoV-2 WA-1 to δ or ο, respectively. CCP antibody levels in the upper 10% of the 108 donations as well as 100% of the post-δ COVID-19/postvaccination units and the hyperimmunoglobulin effectively neutralized all 3 variants. High-titer CCP neutralizes SARS-CoV-2 variants despite no previous donor exposure to the variants., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Polyclonal convalescent plasma may be obtained from donors who have recovered from coronavirus disease 2019 (Covid-19). The efficacy of this plasma in preventing serious complications in outpatients with recent-onset Covid-19 is uncertain., Methods: In this multicenter, double-blind, randomized, controlled trial, we evaluated the efficacy and safety of Covid-19 convalescent plasma, as compared with control plasma, in symptomatic adults (≥18 years of age) who had tested positive for severe acute respiratory syndrome coronavirus 2, regardless of their risk factors for disease progression or vaccination status. Participants were enrolled within 8 days after symptom onset and received a transfusion within 1 day after randomization. The primary outcome was Covid-19-related hospitalization within 28 days after transfusion., Results: Participants were enrolled from June 3, 2020, through October 1, 2021. A total of 1225 participants underwent randomization, and 1181 received a transfusion. In the prespecified modified intention-to-treat analysis that included only participants who received a transfusion, the primary outcome occurred in 17 of 592 participants (2.9%) who received convalescent plasma and 37 of 589 participants (6.3%) who received control plasma (absolute risk reduction, 3.4 percentage points; 95% confidence interval, 1.0 to 5.8; P = 0.005), which corresponded to a relative risk reduction of 54%. Evidence of efficacy in vaccinated participants cannot be inferred from these data because 53 of the 54 participants with Covid-19 who were hospitalized were unvaccinated and 1 participant was partially vaccinated. A total of 16 grade 3 or 4 adverse events (7 in the convalescent-plasma group and 9 in the control-plasma group) occurred in participants who were not hospitalized., Conclusions: In participants with Covid-19, most of whom were unvaccinated, the administration of convalescent plasma within 9 days after the onset of symptoms reduced the risk of disease progression leading to hospitalization. (Funded by the Department of Defense and others; CSSC-004 ClinicalTrials.gov number, NCT04373460.)., (Copyright © 2022 Massachusetts Medical Society.)

Convalescent plasma, collected from donors who have recovered from a pathogen of interest, has been used to treat infectious diseases, particularly in times of outbreak, when alternative therapies were unavailable. The COVID-19 pandemic revived interest in the use of convalescent plasma. Large observational studies and clinical trials that were executed during the pandemic provided insight into how to use convalescent plasma, whereby high levels of antibodies against the pathogen of interest and administration early within the time course of the disease are critical for optimal therapeutic effect. Several studies have shown outpatient administration of COVID-19 convalescent plasma (CCP) to be both safe and effective, preventing clinical progression in patients when administered within the first week of COVID-19. The United States Food and Drug Administration expanded its emergency use authorization (EUA) to allow for the administration of CCP in an outpatient setting in December 2021, at least for immunocompromised patients or those on immunosuppressive therapy. Outpatient transfusion of CCP and infusion of monoclonal antibody therapies for a highly transmissible infectious disease introduces nuanced challenges related to infection prevention. Drawing on our experiences with the clinical and research use of CCP, we describe the logistical considerations and workflow spanning procurement of qualified products, infrastructure, staffing, transfusion, and associated management of adverse events. The purpose of this description is to facilitate the efforts of others intent on establishing outpatient transfusion programs for CCP and other antibody-based therapies., (© 2022 AABB.)

Benchmarks for protective immunity from infection or severe disease after SARS-CoV-2 vaccination are still being defined. Here, we characterized virus neutralizing and ELISA antibody levels, cellular immune responses, and viral variants in 4 separate groups: healthy controls (HCs) weeks (early) or months (late) following vaccination in comparison with symptomatic patients with SARS-CoV-2 after partial or full mRNA vaccination. During the period of the study, most symptomatic breakthrough infections were caused by the SARS-CoV-2 Alpha variant. Neutralizing antibody levels in the HCs were sustained over time against the vaccine parent virus but decreased against the Alpha variant, whereas IgG titers and T cell responses against the parent virus and Alpha variant declined over time. Both partially and fully vaccinated patients with symptomatic infections had lower virus neutralizing antibody levels against the parent virus than the HCs, similar IgG antibody titers, and similar virus-specific T cell responses measured by IFN-γ. Compared with HCs, neutralization activity against the Alpha variant was lower in the partially vaccinated infected patients and tended to be lower in the fully vaccinated infected patients. In this cohort of breakthrough infections, parent virus neutralization was the superior predictor of breakthrough infections with the Alpha variant of SARS-CoV-2.

This study compared the performance of four serology assays for Coronavirus Disease 2019 (COVID-19) and investigated whether COVID-19 disease history correlates with assay performance. Samples were tested at Northshore using the Elecsys Anti-SARS-CoV-2 (Roche Diagnostics), Access SARS-CoV-2 IgG anti-RBD (Beckman Coulter), and LIAISON SARS-CoV-2 S1/S2 IgG (DiaSorin) as well as at Genalyte using Maverick Multi-Antigen Serology Panel. The study included one hundred clinical samples collected before December 2019 and ninety-seven samples collected from convalescent plasma donors originally diagnosed with COVID-19 by PCR. COVID-19 disease history was self-reported by the plasma donors. There was no difference in specificity between the assays tested. Clinical sensitivity of these four tests was 98% (Genalyte), 96% (Roche), 92% (DiaSorin), and 87% (Beckman). The only statistically significant differences in clinical sensitivity was between the Beckman assay and both Genalyte and Roche assays. Convalescent plasma donor characteristics and disease symptoms did not correlate with false negative results from the Beckman and DiaSorin assays. All four tests showed high specificity (100%) and varying sensitivities (89-98%). No correlations between disease history and serology results were observed. The Genalyte Multiplex assay showed as good or better sensitivity to three other previously validated assays with FDA Emergency Use Authorizations., Competing Interests: The authors have read the journal’s policy and have the following competing interests: Procurement and testing of samples collected prior to the year 2020 was paid for by Genalyte, Inc. NorthShore University HealthSystem paid for testing of convalescent plasma donor samples on the three in-house assays, however it was not charged for testing on Genalyte’s assay. Hossein Fakhrai-Rad, Rick Hockett, and Mohammad H. Motamedi are employees of Genalyte, the commercial manufacturer of Genalyte’s SARS-CoV-2 Multi-Antigen Serology Panel. Sean David owns equity in Genalyte. Thomas J. Gniadek, Hong Kee Lee, Jason Kang, Robert C. Benirschke, and Sean David are paid employees of NorthShore University HealthSystem. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The effort to collect convalescent plasma from individuals who recovered from COVID-19 began in earnest during the spring of 2020. Either whole blood or apheresis donations were obtained, the latter yielding higher numbers of units per donor per collection and more frequent collections. The NorthShore University HealthSystem blood donor center purchased 2 Alyx (Fresenius Kabi) apheresis plasma collection devices and quickly implemented them in order to collect COVID-19 convalescent plasma. Apheresis-experienced and inexperienced phlebotomists operated the instruments. Donors were collected >14 days from symptom resolution and all donors were negative by SARS-CoV-2 nasopharyngeal swab. Both internal metrics of performance as well as a post donation survey were used to evaluate the feasibility implementing this collection program. During the first 100 days of the collection program, 650 plasma units were collected. In particular, during the first week of the program, 38 units were collected and distributed to hospitals under the emergency investigational new drug and expanded access program. Fifty-one donors (15%) were deferred due to vital signs out of range or donor screening questions. Thirty-one donors (10%) were deferred due to positive nasopharyngeal swab. Lower than target yield occurred in 16.6% of collections due to donor reactions or flow errors. Donors rated the overall program lower, but not the staff, when they reported symptoms related to collection. In conclusion, a hospital-based apheresis convalescent plasma collection program can be rapidly implemented. Donor reaction rates and vein infiltration rates should be carefully monitored for each phlebotomist., Competing Interests: Declaration of Conflicting Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Technical assistance in preparation of this manuscript was provided by staff of Fresenius Kabi., (© The Author(s) 2021.)

Background: The transfer of passive immunity with convalescent plasma is a promising strategy for treatment and prevention of COVID-19, but donors with a history of nonsevere disease are serologically heterogenous. The relationship between SARS-Cov-2 antigen-binding activity and neutralization activity in this population of donors has not been defined., Study Design and Methods: Convalescent plasma units from 47 individuals with a history of nonsevere COVID-19 were assessed for antigen-binding activity of using three clinical diagnostic serology assays (Beckman, DiaSorin, and Roche) with different SARS-CoV-2 targets. These results were compared with functional neutralization activity using a fluorescent reporter strain of SARS-CoV-2 in a microwell assay., Results: Positive correlations of varying strength (Spearman r = 0.37-0.52) between antigen binding and viral neutralization were identified. Donors age 48 to 75 years had the highest neutralization activity. Units in the highest tertile of binding activity for each assay were enriched (75%-82%) for those with the highest levels of neutralization., Conclusion: The strength of the relationship between antigen-binding activity and neutralization varies depending on the clinical assay used. Units in the highest tertile of binding activity for each assay are predominantly comprised of those with the greatest neutralization activity., (© 2020 AABB.)

Background: Nucleic acid persists after symptom resolution and infectivity for many viral infections via delayed clearance of nucleic acid fragments, non-infectious particles, or transmissible virus. For Coronavirus Disease 2019 (COVID-19), the relationship between nasopharyngeal (NP) swab positivity, the development of antibodies against COVID-19, and clinical history are unclear., Study Design and Methods: Individuals who recovered from COVID-19 and volunteered to donate convalescent plasma (CP) were screened by NP swab PCR, responded to a questionnaire, and were tested for anti-COVID-19 antibodies., Results: A proportion of 11.8% of individuals tested positive for SARS-CoV-2 by NP swab PCR greater than 14 days after the resolution of symptoms of active disease, including one donor who had asymptomatic disease and tested positive by NP swab 41 days after her initial diagnosis. Clinical history did not show a significant correlation with persistence of NP swab positivity. Also, NP swab positivity >14 days from symptom resolution did not correlate with anti-COVID-19 serology results. IgG anti-SARS-CoV-2 spike antibody strength correlated with hospitalization for COVID-19 using two different assays. Total anti-SARS-CoV-2 nucleocapsid antibody strength correlated with time from symptom resolution to sample collection and symptom duration., Conclusions: SARS-CoV-2 nucleic acid is detectable long after the resolution of symptoms in a significant percentage of previously diagnosed individuals, which is important to consider when interpreting PCR swab results. Persistence of PCR positivity does not correlate with antibody strength or symptoms of COVID-19. If anti-spike antibody is used to assess CP potency, individuals who suffered severe COVID-19 disease symptoms may represent better donors., (© 2020 AABB.)

Salmonella has been shown to preferentially colonize solid tumors. It is known that toxicity limits the systemic administration of immunomodulatory cytokines that have a significant anticancer effect. Therefore, we tested a unique cancer treatment strategy comprised of oral delivery of Saltikva, an attenuated strain of Salmonella typhimurium that contain the human gene for interleukin-2. In preclinical experimentation, a significant antitumor effect without toxicity was observed. A dose escalation, single dose, Phase I trial was conducted. Dose escalation (10 to 10) while monitoring for dose limiting toxicity and response was performed. Flow cytometry was conducted to determine the immunologic effect. In total 22 patients were administered Saltikva. Eight patients did not complete the trial. No toxicity or adverse events were observed. There was no survival advantage. Flow cytometry demonstrated an increase in circulating natural killer (NK) cells and NK-T cells when comparing the prestudy period. The results of this phase I dose escalation study show that oral attenuated S. typhimurium containing the human interleukin-2 gene caused no significant toxicities up to doses of 10 colony forming unit. There was no evidence of partial or complete response. All patients had progressive disease and eventually succumbed to their illness. Although no survival advantage was seen in this single dose study, the statistically significant increase in circulating NK and NK-T cell demonstrates an immunologic effect from this treatment regimen and suggest that a multiple dose study should be undertaken.

Objectives: Preanalytical factors, such as hemolysis, affect many components of a test panel. Machine learning can be used to recognize these patterns, alerting clinicians and laboratories to potentially erroneous results. In particular, machine learning might identify which cases of elevated potassium from a point-of-care (POC) basic metabolic panel are likely erroneous., Methods: Plasma potassium concentrations were compared between POC and core laboratory basic metabolic panels to identify falsely elevated POC results. A logistic regression model was created using these labels and the other analytes on the POC panel., Results: This model has high predictive power in classifying POC potassium as falsely elevated or not (area under the curve of 0.995 when applied to the test data set). A rule-in and rule-out approach further improves the model's applicability with a positive predictive value of around 90% and a negative predictive value near 100%., Conclusions: Machine learning has the potential to detect laboratory errors based on the recognition of patterns in commonly requested multianalyte panels. This could be used to alert providers at the POC that a result is suspicious or used to monitor the quality of POC results., (© American Society for Clinical Pathology, 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: When the coronavirus pandemic caused widespread school and business closures in March 2020, blood drives were canceled and the supply of blood decreased suddenly in the United States (US). In response, hospital-based transfusion medicine physicians instituted policies to conserve blood and decrease blood product usage. These efforts were aided by the US Surgeon General recommendation to cancel all elective procedures. Nevertheless, the duration, severity, and impact of the pandemic on the national blood supply was uncertain. Hospitals with in-house donor programs had the opportunity not only to control demand, but also increase supply., Study Design and Methods: A hospital-based blood donor center was rapidly mobilized to increase the supply of in-house collected blood, in order to counteract a sudden but potentially long-term depletion of the national blood supply during a pandemic., Results: Collections increased approximately five-fold above baseline for whole blood units, while apheresis platelet units were maintained at the historical average for the blood donor center. Cancellation of elective procedures showed a modest, but not yet statistically significant decrease in average blood product usage per day, nevertheless the in-house collection rate was sufficient to meet demand., Conclusion: A hospital-based blood donor center can quickly increase collection volumes and capacity in the face of a national emergency or pandemic. The desire to collect units should be balanced with safety concerns, need for sustainability, and blood product demand., (© 2020 AABB.)

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040. 1 ., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Background: Four similar transfusion reactions involving infants were reported in less than 1 year. After transfusion of red blood cells (RBCs) via syringe in the operating room, each patient experienced discolored urine, laboratory evidence of hemolysis, and acute kidney injury. Clerical and serologic investigations were unremarkable. Mechanical hemolysis was considered., Study Design and Methods: Simulated syringe transfusions were performed. Measurements included hematocrit (Hct), free hemoglobin, and visual hemolysis index. Washed and unwashed RBCs were tested with or without a recently introduced one-way valve, using a 24- or 16-gauge intravenous catheter. Constant manual pressure (1.43 ± 0.49 mL/sec) or syringe pump (2 mL/min) was used and a subset was timed., Results: The valve increased hemolysis during manual transfusion using both catheters with washed and unwashed RBCs. With the 24-gauge catheter, the change in Hct was -3.53 ± 0.69% with the valve and 0.22 ± 0.13% without (p < 0.00001). Comparing the individual valves tested, differences in hemolysis were observed (change in Hct, p < 0.0001). During manual transfusion with 24-gauge catheter and unwashed RBCs, the degree of hemolysis was greater when it took longer to transfuse with a valve (change in Hct versus time, r = -0.75, p < 0.0001) compared to a slight increase in hemolysis for samples that took less time to transfuse without a valve (change in Hct versus time, r = 0.58, p = 0.23)., Conclusions: Mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a valve. During rapid manual transfusion with the valve, greater resistance was associated with increased hemolysis., (© 2018 AABB.)

Objective: This case report investigates an unusual hCG result in a woman who is not pregnant., Patient and Methods: A 43-year-old woman was admitted for recurrence of thrombotic thrombocytopenic purpura (TTP) and therapeutic plasma exchange (TPE) was initiated. Prior to transitioning the patient from TPE to immunosuppressive therapy, a serum qualitative hCG test was performed and was positive. Several etiologies for elevated hCG were considered and investigated, including heterophile antibody interference, endogenous hCG from pituitary or malignancy, and exogenous hCG., Results: Retrospective measurement of hCG levels in remnant samples, including a sample obtained prior to TPE initiation, demonstrated that the hCG elevation occurred with plasma administration for TPE. Further investigation with the American Red Cross confirmed that a plasma donor was unknowingly pregnant and in the latter half of the first trimester at the time of donation, when hCG levels peak., Conclusion: In plasma recipients with unexplained hCG elevation, passive transfer of hCG from plasma should be considered in the differential diagnosis. Retrospective measurement of hCG in remnant samples obtained prior to plasma exchange can assist in confirming the source., (Copyright © 2018 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Cell therapy products are often stored and transported between sites. The aim of this study was to determine the effect of storage temperature, solution, and cell concentration on nonmobilized, peripheral blood-derived mononuclear cells (MNCs)., Study Design and Methods: This was a multicenter prospective study involving healthy volunteers who underwent nonmobilized MNC collection by apheresis. Products were processed at local laboratories and concentrated to either 100 × 10 6 or 300 × 10 6 nucleated cells/mL in 5% human serum albumin (HSA) or HypoThermosol FRS (HT; BioLife Solutions). Products were stored at room temperature (RT; 20-25°C) or refrigerated temperatures (2-8°C) with assessment at 0, 24, 48, and 72 hours. NC and MNC concentration, viability, and flow cytometric analysis for CD3, CD4, CD8, CD14, CD19, CD25, and CD56 were measured., Results: Viability decreased over time for all conditions tested. Refrigerated storage preserved viability greater than RT storage, especially for products with a higher cell concentration. RT maintenance with a high cell concentration was associated with a relative loss of CD14- and CD4-positive cells, whereas the concentration of cells positive for other markers tested did not vary. Finally, there was delayed decrease in pH when using HT compared with HSA; however, there was no difference in viability between the two solutions., Conclusion: Low cell concentrations (approx. 100 × 10 6 cells/mL), refrigerated temperatures, and HT storage solution appear to be the optimal conditions for storing nonmobilized, peripheral blood-derived MNC products., (© 2017 AABB.)

Macroautophagy/autophagy is a proteolytic pathway that is involved in both bulk degradation of cytoplasmic proteins as well as in selective degradation of cytoplasmic organelles. Autophagic flux is often defined as a measure of autophagic degradation activity, and many techniques exist to assess autophagic flux. Although these techniques have generated invaluable information about the autophagic system, the quest continues for developing methods that not only enhance sensitivity and provide a means of quantification, but also accurately reflect the dynamic character of the pathway. Based on the theoretical framework of metabolic control analysis, where the autophagosome flux is the quantitative description of the rate a flow along a pathway, here we treat the autophagy system as a multi-step pathway. We describe a single-cell fluorescence live-cell imaging-based approach that allows the autophagosome flux to be accurately measured. This method characterizes autophagy in terms of its complete autophagosome and autolysosome pool size, the autophagosome flux, J, and the transition time, τ, for autophagosomes and autolysosomes at steady state. This approach provides a sensitive quantitative method to measure autophagosome flux, pool sizes and transition time in cells and tissues of clinical relevance., Abbreviations: ATG5/APG5, autophagy-related 5; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; J, flux; MEF, mouse embryonic fibroblast; MTOR, mechanistic target of rapamycin kinase; nA, number of autophagosomes; nAL, number of autolysosomes; nL, number of lysosomes; p-MTOR, phosphorylated mechanistic target of rapamycin kinase; RFP, red fluorescent protein; siRNA, small interfering RNA; τ, transition time; TEM, transmission electron microscopy.

A patient developed disseminated adenovirus infection following bone marrow transplant. TaqMan real-time PCR showed reduced maximum fluorescence in the amplification curve from all plasma samples. Sequencing revealed three single nucleotide mismatches between the TaqMan probe and probe binding region. Real-time PCR with probe matching the isolate sequence showed normal amplification and a higher copy number result., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Salivary blood is known to increase in patients with intraoral mucosal bleeding. Mucosal bleeding is a frequent sequelae of thrombocytopenia, which is typically managed with platelet transfusion. Within the past few years, multiple different types of platelet products have become available, each with potential differences in efficacy. Typically, platelet transfusion efficacy is demonstrated by the increase in platelet count after transfusion. However this approach is complicated by the fact that activated platelets tend to produce lower post-transfusion platelet counts, but may be more efficacious in a bleeding patient. Intraoral blood levels, measured by salivary transferrin, urine dipstick hemoglobin or another method, could be used as an in vivo assay to monitor a patient's response to platelet transfusion and compare different types of platelet products., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Passenger lymphocyte syndrome occurs when donor lymphocytes are transplanted with a solid organ and produce alloantibodies that react with antigens on the recipient's red blood cells (RBCs). Typically, passenger lymphocyte syndrome presents as immunoglobulin G antibody-mediated, extravascular hemolytic anemia with reticulocytosis. Often, the donor was alloimmunized before transplantation., Case Report: A 34-year-old Group O, D+ man with a negative antibody screen received a liver transplant from a Group O, D- donor. Twenty Group O, D+ RBC units were transfused on Postoperative Days (PODs) 0 through 2. On POD 7, the patient developed anemia, a weakly positive antibody screen, and a positive direct antiglobulin test with anti-D in the eluate. After POD 8, a D- transfusion protocol was initiated. Despite laboratory evidence of hemolysis, two initial peripheral blood smears showed no increase in schistocytes or spherocytes, the reticulocyte count was depressed, and a marrow biopsy revealed erythroid hyperplasia. Eventually, anemia resolved after a period of medication non-compliance; however, a positive direct antiglobulin test persisted to the last follow-up date (POD 233)., Results: Other potential causes of aplastic anemia were investigated, but no alternative cause was found. History excluded passive anti-D. D+, LW- cells were reactive, excluding anti-LW. Genotyping showed no evidence of a partial D genotype. Chart review revealed that the liver donor had a history of anti-D. A diagnosis of passenger lymphocyte syndrome was reached., Conclusion: Although antibody-mediated hemolytic anemia has been reported to cause reticulocytopenia in the presence of marrow erythroid hyperplasia, this report of passenger lymphocyte syndrome causing a similar post-transplant anemia in association with reticulocytopenia is noteworthy., (© 2017 AABB.)

Predicting response to checkpoint blockade therapy for lung cancer has largely focused on measuring programmed death-ligand 1 (PD-L1) expression on tumor cells. PD-L1 expression is geographically heterogeneous within many tumors, however, and we questioned whether small tissue samples, such as biopsies, might be sufficiently representative of PD-L1 expression for evaluating this marker in lung cancer tumors. To evaluate the extent of variability of PD-L1 expression in small tissue samples, and how that variability affects accuracy of overall assessment of PD-L1 in lung cancer, we scored immunohistochemical staining for PD-L1 in tissue microarray cores from a series of 79 squamous cell lung cancers and 71 pulmonary adenocarcinomas. Our study found substantial inconsistencies for the percentages of cells staining positive for PD-L1 among different tissue microarray cores in many cases of both adenocarcinoma and squamous cell carcinoma. This variable scoring was seen at both high levels and low levels of PD-L1 expression, and by further evaluation of cases with discordant results on full-face sections to assess geographic distribution of staining, we found that discordant results among different tissue microarray cores reflected geographic variation of PD-L1 expression in those tumors. Moreover, we found that as a result of heterogeneous expression, the sensitivity of a single small tissue sample can be as low as 85% for detecting PD-L1 expression at scoring thresholds commonly used in clinical practice. Based on these studies, we conclude that many cases of lung cancer could be inaccurately or variably scored for PD-L1 expression with a single biopsy sample. Accordingly, lung cancer patients can be inconsistently classified for PD-L1 expression status, particularly when a threshold for the percentage of positive cells is used to determine eligibility for checkpoint blockade therapy.

Objective: Anticentromere antibodies (ACAs) define a subset of primary Sjögren's syndrome (SS) with a unique phenotype, including features of limited cutaneous systemic sclerosis and a lower frequency of anti-SSA/SSB antibodies. We sought to determine whether ACAs are associated with more severe exocrine glandular dysfunction in a large cohort of primary SS subjects., Methods: We performed a cross-sectional analysis of 1,361 subjects with primary SS from the Sjögren's International Collaborative Clinical Alliance Registry, stratified by the presence or absence of ACAs. ACAs were assayed by immunofluorescence staining on HEp-2 cells., Results: ACAs were present in 82 of the 1,361 SS subjects (6%) and were associated with older age, female sex, and lower frequencies of anti-SSA/SSB, rheumatoid factor, and hyperglobulinemia. Among ACA-positive versus ACA-negative subjects, there was a higher frequency of a focus score ≥2 (71% versus 53%; P = 0.002), a higher median focus score (2.8 versus 2.5; P = 0.0440), and greater exocrine gland dysfunction: Schirmer's test value: median 4 versus 5 mm/5 minutes; P = 0.0003, and unstimulated whole saliva (UWS) flow rate: median 0.08 versus 0.37 ml/5 minutes; P < 0.0001. ACA-positive subjects had an increased risk of UWS <0.1 ml/minute (odds ratio [OR] 12.24 [95% confidence interval (95% CI) 4.91-41.02]) and Schirmer's test value <5 mm/5 minutes (OR 2.52 [95% CI 1.50-4.36]) after correcting for age, sex, anti-SSA/SSB, and focus score. Labial gland fibrosis was not different between the 2 groups., Conclusion: In a large international registry of SS, ACA had an independent association with more severe exocrine glandular dysfunction. This dysfunction was associated with more pronounced labial salivary glandular inflammation but not fibrosis., (© 2016, American College of Rheumatology.)

The non-glucose-fermenting Gram-negative bacilli Pseudomonas aeruginosa and Acinetobacter baumannii are increasingly acquiring carbapenem resistance. Given their intrinsic antibiotic resistance, this can cause extremely difficult-to-treat infections. Additionally, resistance gene transfer can occur between Gram-negative species, regardless of their ability to ferment glucose. Thus, the acquisition of carbapenemase genes by these organisms increases the risk of carbapenemase spread in general. Ultimately, infection control practitioners and clinical microbiologists need to work together to determine the risk carried by carbapenem-resistant non-glucose-fermenting Gram-negative bacilli (CR-NF) in their institution and what methods should be considered for surveillance and detection of CR-NF., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We present the cardiac findings from the autopsy of a 28-year-old male with mucopolysaccharidosis VII (MPS VII), also known as Sly Syndrome, whose diagnosis was confirmed by biochemical testing. The patient died a sudden cardiac death. Autopsy showed thickened and stenotic aortic valve leaflets as well as marked concentric intimal thickening of the aorta and muscular arteries. There was left ventricular hypertrophy as well as mild papillary muscle thickening and fusion. Increased colloid iron staining was seen in the small- and medium-sized arteries of the heart and at the intercalated discs. We discuss the patient's premortem echocardiographic and electrocardiographic studies. In addition, we discuss the pathogenesis of MPS VII and review previous literature on its anatomic and pathologic features., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Strategies involving new drug combinations, as well as new uses of existing drugs, are urgently needed to reduce the time required to cure patients with drug-sensitive or multidrug-resistant (MDR) tuberculosis (TB). We compared the sterilizing activity of the standard first-line antitubercular regimen, rifampin-isoniazid-pyrazinamide (RHZ), with that of the novel regimen PA-824-moxifloxacin-pyrazinamide (PaMZ), which is currently being studied in clinical trials (NCT01498419), in the guinea pig model of chronic TB infection, in which animals develop necrotic granulomas histologically resembling their human counterparts. Guinea pigs were aerosol infected with ~2 log10 bacilli of wild-type Mycobacterium tuberculosis H37Rv, and antibiotic treatment was initiated 6 weeks after infection. Separate groups of animals received RHZ, PaMZ, or single or two-drug components of the latter regimen administered at human-equivalent doses 5 days/week for a total of 8 weeks. Relapse rates were assessed 3 months after discontinuation of treatment to determine the sterilizing activity of each combination regimen. PaMZ given at human-equivalent doses was safe and well tolerated for the entire treatment period and rendered guinea pig lungs culture negative more rapidly than RHZ did. After 1 month of treatment, 80% and 50% of animals in the RHZ and PaMZ groups, respectively, had lung culture-positive relapse. Both combination regimens prevented microbiological relapse when administered for a total of 2 months. Our data support the use of PaMZ as a novel isoniazid- and rifamycin-sparing regimen suitable for treatment of both drug-sensitive TB and MDR-TB.

Recombinant techniques for expressing heterologous proteins and sugars on the surface of bacteria have been known since the 1980s, and have proven useful in a variety of settings from biocatalysis to vaccinology. The past decade has also seen the emergence of novel methods that allow modification of bacterial surfaces with small non-biological compounds. Such technologies enable researchers to harness the unique properties of synthetic materials on a live bacterial platform, opening the door to an exciting new set of applications. Here we review strategies for bacterial surface display and describe how they have been applied thus far. We believe that chemical surface display holds great potential for advancing research in basic bacteriology and applied fields of biotechnology and biomedicine., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Golgins are coiled-coil proteins that play a key role in the regulation of Golgi architecture and function. Giantin, the largest golgin in mammals, forms a complex with p115, rab1, GM130, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), thereby facilitating vesicle tethering and fusion processes around the Golgi apparatus. Treatment with the microtubule destabilizing drug nocodazole transforms the Golgi ribbon into individual Golgi stacks. Here we show that siRNA-mediated depletion of giantin resulted in more dispersed Golgi stacks after nocodazole treatment than by control treatment, without changing the average cisternal length. Furthermore, depletion of giantin caused an increase in cargo transport that was associated with altered cell surface protein glycosylation. Drosophila S2 cells are known to have dispersed Golgi stacks and no giantin homolog. The exogenous expression of mammalian giantin cDNA in S2 cells resulted in clustered Golgi stacks, similar to the Golgi ribbon in mammalian cells. These results suggest that the spatial organization of the Golgi ribbon is mediated by giantin, which also plays a role in cargo transport and sugar modifications.

Current research in cell biology frequently uses light microscopy to study intracellular organelles. To segment and count organelles, most investigators have used a global thresholding method, which relies on homogeneous background intensity values within a cell. Because this is not always the case, we developed WatershedCounting3D, a program that uses a modified watershed algorithm to more accurately identify intracellular structures from confocal image data, even in the presence of an inhomogeneous background. We give examples of segmenting and counting endoplasmic reticulum exit sites and the Golgi apparatus.

Objective: We describe a new tool to facilitate the creation and interactive display of stacks of images for scientific and educational presentations using Microsoft PowerPoint., Conclusion: This new tool allows interactive scrolling through stacks of images. After a presentation is created, all elements required to display the stacks of images are embedded in the PowerPoint file. These presentations can be run on any computer (Macintosh or IBM-compatible versions) using PowerPoint with no other preinstalled software, making this tool useful for scientific and educational presentations at national and international meetings.