Significantly Decreased Mortality in a Large Cohort of Coronavirus Disease 2019 (COVID-19) Patients Transfused Early with Convalescent Plasma Containing High-Titer Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike Protein IgG.

Coronavirus disease 2019 (COVID-19) convalescent plasma has emerged as a promising therapy and has been granted Emergency Use Authorization by the US Food and Drug Administration for hospitalized COVID-19 patients. We recently reported results from interim analysis of a propensity score-matched study suggesting that early treatment of COVID-19 patients with convalescent plasma containing high-titer anti-spike protein receptor binding domain (RBD) IgG significantly decreases mortality. We herein present results from a 60-day follow-up of a cohort of 351 transfused hospitalized patients. Prospective determination of enzyme-linked immunosorbent assay anti-RBD IgG titer facilitated selection and transfusion of the highest titer units available. Retrospective analysis by the Ortho VITROS IgG assay revealed a median signal/cutoff ratio of 24.0 for transfused units, a value far exceeding the recent US Food and Drug Administration-required cutoff of 12.0 for designation of high-titer convalescent plasma. With respect to altering mortality, our analysis identified an optimal window of 44 hours after hospitalization for transfusing COVID-19 patients with high-titer convalescent plasma. In the aggregate, the analysis confirms and extends our previous preliminary finding that transfusion of COVID-19 patients soon after hospitalization with high-titer anti-spike protein RBD IgG present in convalescent plasma significantly reduces mortality.

Treatment of Coronavirus Disease 2019 Patients with Convalescent Plasma Reveals a Signal of Significantly Decreased Mortality.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and proven treatments are limited. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 is among many approaches being studied as potentially efficacious therapy. We are conducting a prospective, propensity score-matched study assessing the efficacy of COVID-19 convalescent plasma transfusion versus standard of care as treatment for severe and/or critical COVID-19. We present herein the results of an interim analysis of 316 patients enrolled at Houston Methodist hospitals from March 28 to July 6, 2020. Of the 316 transfused patients, 136 met a 28-day outcome and were matched to 251 non-transfused control COVID-19 patients. Matching criteria included age, sex, body mass index, comorbidities, and baseline ventilation requirement 48 hours from admission, and in a second matching analysis, ventilation status at day 0. Variability in the timing of transfusion relative to admission and titer of antibodies of plasma transfused allowed for analysis in specific matched cohorts. The analysis showed a significant reduction (P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350. These data suggest that treatment of COVID-19 with high anti-receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients.

Treatment of Coronavirus Disease 2019 (COVID-19) Patients with Convalescent Plasma.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n = 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

A Simulation Course Focusing on Forensic Evidence Collection Improves Pediatric Knowledge and Standardizes Curriculum for Child Abuse.

OBJECTIVES: Our hypothesis was that pediatric residents and medical students who participated in a structured forensic evidence collection course would have improved knowledge of prepubertal evidence collection practices and pubertal genital anatomy. METHODS: The course curriculum included a forensic evidence collection video created by the sexual assault nurse examiner directors. After watching the video, the participants simulated forensic evidence collection using forensic evidence collection kits and chain of evidence protocols in a hybrid simulation setting under the supervision of a pediatric sexual assault nurse examiner. The participants completed a multiple-choice test and a fill-in-the-blank anatomical diagram test before and after the course. RESULTS: Of an eligible 48 participants, 42 completed the course; therefore, our participant response rate was 87.5%. There was significant improvement in knowledge, with an average pretest score of 62% ± 20% and the average posttest score of 86% ± 9% (P < 0.001). Qualitative evaluations were overwhelmingly positive, with consistent scoring of 6/6 in a 6-point agree scale. Learning themes, which emerged from open-ended questions on the evaluations, included knowledge gained on evidence collection processes (n = 26), how to appropriately interact with abused patients (n = 8), hands-on nature of the experience and the benefits of walking through the examination (n = 7), and pubertal genital anatomy knowledge (n = 3). Participants suggested that more instruction on anatomy would be helpful. CONCLUSIONS: We found that pediatric residents' and medical students' knowledge of pediatric sexual abuse may be improved with a short simulation course focusing on forensic evidence collection.

Analytical performance of QMS everolimus assay on ortho Vitros 5,1 FS fusion analyzer: measuring everolimus trough levels for solid organ transplant recipients.

BACKGROUND: Everolimus has recently been approved by Food and Drug Administration for graft maintenance in liver transplant recipients. This drug has a narrow therapeutic index and benefits from close blood level monitoring. Currently, in the United States, the Thermo Fisher Scientific Quantitative Microsphere System (QMS) Everolimus Immunoassay is the only Food and Drug Administration-cleared immunoassay for monitoring everolimus in renal transplant recipients. However, studies on this assay adapted to the Ortho Vitros 5,1 FS chemistry analyzer have not been published, and data of this assay applied to monitoring drug levels in liver transplant recipients are limited. Here, the authors evaluated and validated the QMS everolimus assay on the Vitros analyzer and its application to supporting the immunosuppressant management of mainly liver transplant recipients. METHODS: The analysis was performed according to the QMS assay package insert. The method was compared with a liquid chromatography-tandem mass spectrometry method from a reference laboratory using a total of 34 samples from 1 double lung and liver, 8 liver, and 3 kidney recipients. The method comparison was assessed by Deming regression. Proficiency test materials issued by Everolimus TDM Proficiency Support Program were tested and compared with the peer group results of using the QMS kits. RESULTS: The assay was linear in the range of 0.75-20.0 ng/mL. Limit of detection was 0.70 ng/mL and lower limit of quantitation was 0.75 ng/mL. Within-day and between-day (20 days) coefficients of variation were between 3.1% and 16.5% at mean levels of 5.3, 12.0, and 17.2 ng/mL, respectively. We obtained a Deming regression of y = 1.271 - 0.666 (r = 0.880) when comparing with the liquid chromatography-tandem mass spectrometry method. CONCLUSIONS: The authors concluded that the analytical performance of the QMS everolimus immunoassay by the Vitros 5,1 FS analyzer was satisfactory for monitoring drug levels of solid organ transplant patients.

The Reemergence of Seasonal Respiratory Viruses in Houston, Texas, after Relaxing COVID-19 Restrictions.

Measures intended to limit the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus at the start of the coronavirus disease 2019 (COVID-19) pandemic resulted in a rapid decrease in other respiratory pathogens. Herein, we describe the trends of respiratory pathogens in a major metropolitan health care system central microbiology reference laboratory before and during the COVID-19 pandemic, with attention to when COVID-19 mitigation measures were implemented and relaxed. During the initial lockdown period, COVID-19 was the primary respiratory pathogen detected by multiplex respiratory panels. As COVID-19 containment measures were relaxed, the first non-COVID respiratory viruses to return to prepandemic levels were members of the rhinovirus/enterovirus family. After the complete removal of COVID-19 precautions at the state level, including an end to mask mandates, we observed the robust return of seasonal coronaviruses, parainfluenza virus, and respiratory syncytial virus. Inasmuch as COVID-19 has dominated the landscape of respiratory infections since early 2020, it is important for clinicians to recognize that the return of non-COVID respiratory pathogens may be rapid and significant when COVID-19 containment measures are removed. IMPORTANCE We describe the return of non-COVID respiratory viruses after the removal of COVID-19 mitigation measures. It is important for the public and physicians to recognize that, after months of COVID-19 being the primary driver of respiratory infection, more typical seasonal respiratory illnesses have returned, and this return is out of the normal season for some of these pathogens. Thus, clinicians and the public must now consider both COVID-19 and other respiratory illnesses when a patient presents with symptomatic respiratory illness.

Limited window for donation of convalescent plasma with high live-virus neutralizing antibody titers for COVID-19 immunotherapy.

Millions of individuals who have recovered from SARS-CoV-2 infection may be eligible to participate in convalescent plasma donor programs, yet the optimal window for donating high neutralizing titer convalescent plasma for COVID-19 immunotherapy remains unknown. Here we studied the response trajectories of antibodies directed to the SARS-CoV-2 surface spike glycoprotein and in vitro SARS-CoV-2 live virus neutralizing titers (VN) in 175 convalescent donors longitudinally sampled for up to 142 days post onset of symptoms (DPO). We observed robust IgM, IgG, and viral neutralization responses to SARS-CoV-2 that persist, in the aggregate, for at least 100 DPO. However, there is a notable decline in VN titers ≥160 for convalescent plasma therapy, starting 60 DPO. The results also show that individuals 30 years of age or younger have significantly lower VN, IgG and IgM antibody titers than those in the older age groups; and individuals with greater disease severity also have significantly higher IgM and IgG antibody titers. Taken together, these findings define the optimal window for donating convalescent plasma useful for immunotherapy of COVID-19 patients and reveal important predictors of an ideal plasma donor.

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

We sequenced the genomes of 5,085 SARS-CoV-2 strains causing two COVID-19 disease waves in metropolitan Houston, Texas, an ethnically diverse region with seven million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston, and an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotypes and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein - the primary target of global vaccine efforts - are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR30022. Our study is the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves, and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.

Convalescent plasma anti-SARS-CoV-2 spike protein ectodomain and receptor-binding domain IgG correlate with virus neutralization.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the urgent need for assays that detect protective levels of neutralizing antibodies. We studied the relationship among anti-spike ectodomain (anti-ECD), anti-receptor-binding domain (anti-RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by 2 in vitro assays using convalescent plasma samples from 68 patients with COVID-19. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers and in vitro VN titers. The probability of a VN titer of ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment, was ≥80% when anti-RBD or anti-ECD titers were ≥1:1350. Of all donors, 37% lacked VN titers of ≥160. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease VN or IgG titers. Analysis of 2814 asymptomatic adults found 73 individuals with anti-ECD IgG titers of ≥1:50 and strong positive correlation with anti-RBD and VN titers. Fourteen of these individuals had VN titers of ≥1:160, and all of them had anti-RBD titers of ≥1:1350. We conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titers of ≥1:1350 may provide critical information about protection against COVID-19 disease.

Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area.

We sequenced the genomes of 5,085 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains causing two coronavirus disease 2019 (COVID-19) disease waves in metropolitan Houston, TX, an ethnically diverse region with 7 million residents. The genomes were from viruses recovered in the earliest recognized phase of the pandemic in Houston and from viruses recovered in an ongoing massive second wave of infections. The virus was originally introduced into Houston many times independently. Virtually all strains in the second wave have a Gly614 amino acid replacement in the spike protein, a polymorphism that has been linked to increased transmission and infectivity. Patients infected with the Gly614 variant strains had significantly higher virus loads in the nasopharynx on initial diagnosis. We found little evidence of a significant relationship between virus genotype and altered virulence, stressing the linkage between disease severity, underlying medical conditions, and host genetics. Some regions of the spike protein-the primary target of global vaccine efforts-are replete with amino acid replacements, perhaps indicating the action of selection. We exploited the genomic data to generate defined single amino acid replacements in the receptor binding domain of spike protein that, importantly, produced decreased recognition by the neutralizing monoclonal antibody CR3022. Our report represents the first analysis of the molecular architecture of SARS-CoV-2 in two infection waves in a major metropolitan region. The findings will help us to understand the origin, composition, and trajectory of future infection waves and the potential effect of the host immune response and therapeutic maneuvers on SARS-CoV-2 evolution.IMPORTANCE There is concern about second and subsequent waves of COVID-19 caused by the SARS-CoV-2 coronavirus occurring in communities globally that had an initial disease wave. Metropolitan Houston, TX, with a population of 7 million, is experiencing a massive second disease wave that began in late May 2020. To understand SARS-CoV-2 molecular population genomic architecture and evolution and the relationship between virus genotypes and patient features, we sequenced the genomes of 5,085 SARS-CoV-2 strains from these two waves. Our report provides the first molecular characterization of SARS-CoV-2 strains causing two distinct COVID-19 disease waves.

Relationship between Anti-Spike Protein Antibody Titers and SARS-CoV-2 In Vitro Virus Neutralization in Convalescent Plasma.

Newly emerged pathogens such as SARS-CoV-2 highlight the urgent need for assays that detect levels of neutralizing antibodies that may be protective. We studied the relationship between anti-spike ectodomain (ECD) and anti-receptor binding domain (RBD) IgG titers, and SARS-CoV-2 virus neutralization (VN) titers generated by two different in vitro assays using convalescent plasma samples obtained from 68 COVID-19 patients, including 13 who donated plasma multiple times. Only 23% (16/68) of donors had been hospitalized. We also studied 16 samples from subjects found to have anti-spike protein IgG during surveillance screening of asymptomatic individuals. We report a strong positive correlation between both plasma anti-RBD and anti-ECD IgG titers, and in vitro VN titer. Anti-RBD plasma IgG correlated slightly better than anti-ECD IgG titer with VN titer. The probability of a VN titer ≥160 was 80% or greater with anti-RBD or anti-ECD titers of ≥1:1350. Thirty-seven percent (25/68) of convalescent plasma donors lacked VN titers ≥160, the FDA-recommended level for convalescent plasma used for COVID-19 treatment. Dyspnea, hospitalization, and disease severity were significantly associated with higher VN titer. Frequent donation of convalescent plasma did not significantly decrease either VN or IgG titers. Analysis of 2,814 asymptomatic adults found 27 individuals with anti-RBD or anti-ECD IgG titers of ≥1:1350, and evidence of VN ≥1:160. Taken together, we conclude that anti-RBD or anti-ECD IgG titers can serve as a surrogate for VN titers to identify suitable plasma donors. Plasma anti-RBD or anti-ECD titer of ≥1:1350 may provide critical information about protection against COVID-19 disease.

Treatment of COVID-19 Patients with Convalescent Plasma in Houston, Texas.

BACKGROUND: COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for more than 100 years. METHODS: Patients (n=25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28 to April 14, 2020. Patients were transfused with convalescent plasma obtained from donors with confirmed SARS-CoV-2 infection and had been symptom free for 14 days. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 post-transfusion. Clinical improvement was assessed based on a modified World Health Organization 6-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. RESULTS: At baseline, all patients were receiving supportive care, including anti-inflammatory and anti-viral treatments, and all patients were on oxygen support. At day 7 post-transfusion with convalescent plasma, nine patients had at least a 1-point improvement in clinical scale, and seven of those were discharged. By day 14 post-transfusion, 19 (76%) patients had at least a 1-point improvement in clinical status and 11 were discharged. No adverse events as a result of plasma transfusion were observed. The whole genome sequencing data did not identify a strain genotype-disease severity correlation. CONCLUSIONS: The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease. Randomized, controlled trials are needed to determine its efficacy.

Sedation after intubation using etomidate and a long-acting neuromuscular blocker.

BACKGROUND: Etomidate is an imidazole hypnotic which is commonly used by emergency medicine physicians during rapid sequence intubation. Etomidate's duration of action is significantly shorter than that of commonly used long-acting paralytic medications (3-12 minutes vs 25-73 minutes). If additional sedative medications are not administered in the paralyzed patient before the conclusion of etomidate's duration of action, patients are at risk for experiencing paralysis without adequate sedation. OBJECTIVE: To evaluate the frequency of the administration of additional sedation in pediatric emergency department patients undergoing endotracheal intubation with etomidate and a long-acting paralytic agent. METHODS: This study was a retrospective review of pediatric patients undergoing endotracheal intubation in a tertiary pediatric emergency department between July 2001 and December 2005. All patients intubated with etomidate and rocuronium or vecuronium were eligible for inclusion; patients with seizures were excluded. Data elements included the following: demographic variables, presenting complaint, intubation indication, medications used, time from etomidate administration to the administration of an additional sedative, Glasgow Coma Scale (GCS) score, and patient disposition. RESULTS: During the study period, 276 pediatric intubations were reviewed with 104 patients receiving etomidate and rocuronium or vecuronium. Twenty cases were excluded, 15 cases with documented seizures and 5 incomplete/missing charts. Eighty-four records were included in the final analysis. The mean age is 84 +/- 65 months; 62 (73.8%) patients were male; the mean GCS was 8.44 +/- 3.9, with a median GCS of 8 (interquartile range 6,11), and 41 (48.8%) of patients presented with blunt trauma. The mean time from etomidate to the administration of additional sedation was 46 +/- 49 minutes. Eleven (13.1%) patients received no additional sedative after etomidate administration, whereas only 20 (23.8%) patients were given a sedative within 15 minutes of the administration of etomidate. Fifty-three (63.1%) patients received an additional sedative more than 15 minutes after the administration of etomidate. CONCLUSIONS: A significant proportion of pediatric patients receiving etomidate and rocuronium or vecuronium during endotracheal intubation are likely experiencing ongoing paralysis without adequate sedation. Emergency medicine physicians should be cognizant of this when using these medications for facilitating intubation.

Metabolomic biomarkers are associated with mortality in patients with cirrhosis caused by primary biliary cholangitis or primary sclerosing cholangitis.

AIM: To assess the ability of signature metabolites alone, or in combination with the model for end-stage liver disease-Na (MELD-Na) score to predict mortality in patients with cirrhosis caused by primary biliary cholangitis or primary sclerosing cholangitis. MATERIALS & METHODS: Plasma metabolites were detected using ultrahigh-performance liquid chromatography/tandem mass spectrometry in 39 patients with cirrhosis caused by primary biliary cholangitis or primary sclerosing cholangitis. Mortality was predicted using Cox proportional hazards regression and time-dependent receiver operating characteristic curve analyses. RESULTS: The top five metabolites with significantly greater accuracy than the MELD-Na score (area under the receiver operating characteristic curve [AUROC] = 0.7591) to predict 1-year mortality were myo-inositol (AUROC = 0.9537), N-acetylputrescine (AUROC = 0.9018), trans-aconitate (AUROC = 0.8880), erythronate (AUROC = 0.8345) and N6-carbamoylthreonyladenosine (AUROC = 0.8055). Several combined MELD-Na-metabolite models increased the accuracy of predicted 1-year mortality substantially (AUROC increased from 0.7591 up to 0.9392). CONCLUSION: Plasma metabolites have the potential to enhance the accuracy of mortality predictions, minimize underestimates of mortality in patients with cirrhosis and low MELD-Na scores, and promote equitable allocation of donor livers.

The Prevalence and Role of Hemoglobin Variants in Biometric Screening of a Multiethnic Population: One Large Health System's Experience.

OBJECTIVES: To characterize and quantitate hemoglobin (Hb) variants discovered during biometric hemoglobin A1c (HbA1c) analyses in a large multiethnic population with a focus on the effect of variants on testing method and results. METHODS: In total, 13,913 individuals had their HbA1c measured via ion-exchange high-performance liquid chromatography. Samples that had a variant Hb detected or HbF fraction more than 25% underwent variant Hb characterization and confirmation by gel electrophoresis. RBC indices were also evaluated for possible concomitant thalassemia. RESULTS: Of the 13,913 individuals evaluated, 524 (3.77%) had an Hb variant. The prevalence of each variant was as follows: HbS trait (n = 396, 2.85%), HbSS disease (n = 4, 0.03%), HbC trait (n = 85, 0.61%), HbCC disease (n = 2, 0.01%), HbSC disease (n = 5, 0.04%), HbE trait (n = 18, 0.13%), HbD or G trait (n = 9, 0.06%), HbS ß-thalassemia + disease (n = 1, 0.01%), hereditary persistence of HbF (n = 2, 0.01%), and HbMontgomery trait (n = 1, 0.01%). Concomitant α-thalassemia was detected in 20 (3.82%) of the 524 individuals with an Hb variant. CONCLUSIONS: This study represents one of the largest epidemiologic investigations into the prevalence of Hb variants in a North American metropolitan, multiethnic workforce and their dependents and reinforces the importance of method selection in populations with Hb variants.

Nanoplasmonic Quantification of Tumor-derived Extracellular Vesicles in Plasma Microsamples for Diagnosis and Treatment Monitoring.

Tumour-derived extracellular vesicles (EVs) are of increasing interest as a resource of diagnostic biomarkers. However, most EV assays require large samples, are time-consuming, low-throughput and costly, and thus impractical for clinical use. Here, we describe a rapid, ultrasensitive and inexpensive nanoplasmon-enhanced scattering (nPES) assay that directly quantifies tumor-derived EVs from as little as 1 µL of plasma. The assay uses the binding of antibody-conjugated gold nanospheres and nanorods to EVs captured by EV-specific antibodies on a sensor chip to produce a local plasmon effect that enhances tumour-derived EV detection sensitivity and specificity. We identified a pancreatic cancer EV biomarker, ephrin type-A receptor 2 (EphA2), and demonstrate that an nPES assay for EphA2-EVs distinguishes pancreatic cancer patients from pancreatitis patients and healthy subjects. EphA2-EVs were also informative in staging tumour progression and in detecting early responses to neoadjuvant therapy, with better performance than a conventional enzyme-linked immunosorbent assay. The nPES assay can be easily refined for clinical use, and readily adapted for diagnosis and monitoring of other conditions with disease-specific EV biomarkers.

The presence of anti-HLA donor-specific antibodies in lung allograft recipients does not correlate with C4d immunofluorescence in transbronchial biopsy specimens.

CONTEXT: C4d immunofluorescence (IF) is a surrogate for development of donor-specific antibodies (DSAs) against human leukocyte antigen (HLA) class I and II antigens in kidney and heart biopsy specimens for monitoring of antibody-mediated (humoral) allograft rejection (AMR). Use of C4d IF in monitoring of lung allografts has shown conflicting results. OBJECTIVE: To determine if C4d IF can be used as a reliable marker for AMR and if it correlates with the presence of DSAs and histologic findings on biopsy. DESIGN: All transbronchial biopsies in lung allograft recipients, performed at our institution in a 3-year period, were reviewed. A cohort of 92 patients with 110 corresponding biopsies met the inclusion criteria of (1) having a resulted DSA within 2 weeks of biopsy and (2) having C4d immunofluorescence studies performed and confirmed. RESULTS: Twenty-nine patients (31.5%) were positive for DSAs and 63 patients (68.5%) did not develop DSAs. Positive C4d capillary IF was seen in 18 of 110 total biopsy specimens (16.4%). Eight of these biopsy samples were from patients positive for DSAs and 10 were from patients negative for DSAs. The correlation coefficient between the presence of DSAs and C4d IF was 0.1628 (P = .09). CONCLUSIONS: A significant proportion of DSA-positive patients had negative C4d IF results and frequently have no histologic changes on biopsy specimens. DSA-negative patients can be positive for C4d and may show the same histologic changes as reported for DSA-positive patients. Diagnosis of AMR in lung may require a collaborative approach combining clinical data, DSA status, and histology.

In utero drugs of abuse exposure testing for newborn twins.

This report describes testing of a case of in utero drugs of abuse exposure in which discordant results were seen between urine and meconium, and between twin meconium samples. The discordance between urine and meconium could be explained by the differences in detection window, threshold concentration and screening technology, and the discordance between dizygotic twin meconium samples could be explained by the differences in drug diffusion and placental and fetal biotransformation of drugs. The meconium sample of one twin screened negative for benzodiazepines was reported positive in the confirmation assay with higher sensitivity and a lower cut-off concentration. Negative screening results of drugs of abuse should be interpreted with caution, taking into account matrix type, reactivity of drugs in the assay and cut-off concentration. If screening results are inconsistent with each other or with the clinical scenario, confirmation testing using more sensitive and specific methods with lower cut-offs is warranted.