Assessing the association between antibody status and symptoms of long COVID: A multisite study.

The association between SARS-CoV-2 humoral immunity and post-acute sequelae of COVID-19 (long COVID) remains uncertain. The objective of this population-based cohort study was to assess the association between SARS-CoV-2 seropositivity and symptoms consistent with long COVID. English and Spanish-speaking members ≥ 18 years old with SARS-CoV-2 serologic testing conducted prior to August 2021 were recruited from Kaiser Permanente Southern California and Kaiser Permanente Colorado. Between November 2021 and April 2022, participants completed a survey assessing symptoms, physical health, mental health, and cognitive function consistent with long COVID. Survey results were linked to SARS-CoV-2 antibody (Ab) and viral (RNA) lab results in electronic health records. Weighted descriptive analyses were generated for five mutually exclusive patient groups: (1) +Ab/+RNA; (2) +Ab/- or missing RNA; (3) -Ab/+RNA; (4a) -Ab/-RNA reporting no prior infection; and (4b) -Ab/-RNA reporting prior infection. The proportions reporting symptoms between the +Ab/+RNA and -Ab/+RNA groups were compared, adjusted for covariates. Among 3,946 participants, the mean age was 52.1 years old (SD 15.6), 68.3% were female, 28.4% were Hispanic, and the serologic testing occurred a median of 15 months prior (IQR = 12-18). Three quarters (74.5%) reported having had COVID-19. Among people with laboratory-confirmed COVID-19, there was no association between antibody positivity (+Ab/+RNA vs. -Ab/+RNA) and any symptoms, physical health, mental health, or cognitive function. As expected, physical health, cognitive function, and fatigue were worse, and palpitations and headaches limiting the ability to work were more prevalent among people with laboratory-confirmed prior infection and positive serology (+Ab/+RNA) compared to those without reported or confirmed prior infection and negative serology (-Ab/-RNA/no reported COVID-19). Among people with laboratory-confirmed COVID-19, SARS-CoV-2 serology from practice settings were not associated with long COVID symptoms and health status suggesting limited utility of serology testing for long COVID.

Lessons learned from the use of COVID-19 convalescent plasma at Kaiser Permanente.

Background: In April 2020, the Mayo Clinic helped establish the US Food and Drug Administration Expanded Access Protocol for COVID-19 (coronavirus disease 2019) convalescent plasma (CCP). The effectiveness of CCP in the published literature is contradictory because some retrospective studies showed benefit in reducing mortality and severe illness, whereas prospective randomized controlled trials demonstrated no benefit of CCP. Objectives: To discuss (1) the implementation of CCP across Kaiser Permanente Southern California between April 2020 and April 2021, (2) retrospective multivariable analysis of 2,831 patients with COVID-19 who were transfused with CCP compared with 18,475 patients with COVID-19 who did not receive CCP, (3) how to reconcile contradictory published data regarding the efficacy of CCP, and (4) guidance regarding the future use of convalescent plasma in a large community hospital setting. Methods: Multivariable analysis was controlled for demographic characteristics, level of oxygen delivery, intensive care unit stay, selected laboratory findings, and other concurrent treatment-related variables. Tubing segments from 151 CCP units transfused between October 2020 and April 2021 were retrospectively tested for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike protein receptor-binding domain IgG. Multivariable analysis showed that CCP transfusion did not affect mortality rates at 30 days and 5 months (odds ratio, 1.04, 95% CI, 0.87-1.25, and hazard ratio, 1.05, 95% CI, 0.93-1.19). Conclusions: If convalescent plasma is offered as a therapeutic in a future viral pandemic, we recommend (1) transfusing only those patients who are negative for neutralizing antibodies, (2) transfusing very early during the disease course, (3) only using convalescent plasma with known levels of neutralizing antibodies, or (4) alternatively providing fractionated hyperimmune globulin.

Elevated von Willebrand Factor Antigen Is an Early Predictor of Mortality and Prolonged Length of Stay for Coronavirus Disease 2019 (COVID-19) Inpatients.

CONTEXT.­: Coagulation factor and endothelial injury marker, von Willebrand factor antigen (vWF:Ag), is elevated in coronavirus disease 2019 (COVID-19). OBJECTIVE.­: To assess the prognostic value of vWF:Ag for COVID-19 inpatients. DESIGN.­: Citrated plasma samples collected from COVID-19 inpatients for D-dimer measurement were tested for vWF:Ag. Measurements of vWF:Ag and common acute-phase reactants were correlated with clinical outcomes and length of stay (LOS). RESULTS.­: We included 333 samples from a diverse group of 120 COVID-19 inpatients. There was a clear association of higher peak measurements of vWF:Ag and other acute-phase reactants with adverse clinical outcomes. Peak vWF:Ag >300% was associated with a 5-fold increased risk of death (odds ratio [OR], 5.08; P < .001) and a 30-fold increased risk of prolonged (>4 days) LOS (OR, 29.65; P = .001). Peak D-dimer >3.8 fibrinogen equivalent units (FEUs) mg/L was associated with a 15-fold increase in risk of death (OR, 14.73; P < .001) and a 5-fold increased risk of prolonged LOS (OR, 4.55; P = .02). Using the earliest paired measurements of vWF:Ag and D-dimer from each patient and the same cutoffs, vWF:Ag was associated with a 3.5-fold increase in risk of death (OR, 3.54; P = .004) and a 20-fold risk of prolonged LOS (OR, 20.19; P = .004). Yet D-dimer was not significantly associated with either death (OR, 1.9; P = .29) or prolonged LOS (OR, 1.02; P = .98). CONCLUSIONS.­: Both peak and early postadmission vWF:Ag >300% were highly predictive of death and prolonged LOS among COVID-19 inpatients. Measurement of vWF:Ag may prove a valuable tool to guide escalation of COVID-19 treatment, particularly anticoagulation.

Effect of Using an Age-adjusted D-dimer to Assess for Pulmonary Embolism in Community Emergency Departments.

BACKGROUND: The objective of this study was to evaluate the effect of changing the laboratory-reported D-dimer reference intervals to age-adjusted reference intervals on the use of advanced chest imaging and 30-day adverse events among emergency department (ED) encounters. METHODS: A retrospective interrupted time-series analysis of ED encounters for patients > 50 years evaluated for suspected pulmonary embolism (PE) from April 2014 to April 2016. The primary outcome was use of advanced diagnostic imaging, and the secondary outcome was 30-day mortality or PE diagnosis. Secondary analyses also quantified delayed PE diagnoses pre- and postintervention. A generalized estimating equation segmented logistic regression model, adjusting for patient and facility characteristics, was used to determine changes in odds of diagnostic imaging and 30-day mortality or PE diagnoses. RESULTS: A total of 10,534 (5,153 pre- and 5,381 postimplementation) ED encounters were included. Advanced imaging was obtained in 35.9% of pre- versus 33% of postimplementation encounters. Age-adjusted D-dimer (AADD) showed a small and nonsignificant decrease in month-to-month trends of advanced chest imaging postimplementation (odds ratio [OR] = 0.98, 95% confidence interval [CI] = 0.96 to 1.00). Use of advanced imaging in patients with D-dimer values lower than 500 ng/mL fibrinogen-equivalent units (FEU) was similar in the preintervention (5.8%) and postintervention (6.8%) periods. However, imaging was obtained in 30% of patients postintervention with a D-dimer result less than AADD reference interval , but more than the historical 500 ng/mL FEU reference interval. Implementing an AADD threshold demonstrated no change in the rate of 30-day adverse events (missed PE or mortality). CONCLUSION: Changing the laboratory-reported D-dimer reference intervals for evaluation of PE was not associated with reduction in advanced chest imaging and did not increase 30-day adverse events. However, there was substantial noncompliance with the age-adjusted reference intervals in the postintervention period likely blunting the impact of this intervention.

Age-Dependent Hemoglobin A1c Therapeutic Targets Reduce Diabetic Medication Changes in the Elderly.

OBJECTIVE: To assess whether implementation of age-dependent therapeutic targets for high hemoglobin A1c (HbA1c) changed clinicians' ordering of diabetes medications for older adults. BACKGROUND: In 2016, Kaiser Permanente Southern California (KPSC) changed the therapeutic targets for alerting clinicians about high HbA1c results in the electronic health record, KP HealthConnect (KPHC). Previously, all HbA1c results ≥7.0 percent were flagged as high in adult patients with diabetes. Starting in 2016, HbA1c therapeutic targets were relaxed to <7.5 percent for patients age 65 to 75, and to <8.0 percent for patients over age 75 to reduce treatment intensity and adverse events. METHODS: This retrospective analysis used logistic regression models to calculate the change in odds of a medication change following an HbA1c result after age-dependent HbA1c flags were introduced. RESULTS: The odds of medication change decreased among patients whose HbA1c targets were relaxed: Odds Ratio (OR) 0.72 (95 percent CI 0.67-0.76) for patients age 65-75 and HbA1c 7.0 percent-7.5 percent; OR 0.72 (95 percent CI 0.65-0.80) for patients over age 75 and HbA1c 7.0 percent-7.5 percent; and OR 0.67 (95 percent CI 0.61-0.75) for patients over age 75 and HbA1c 7.5 percent-8.0 percent. In the age and HbA1c ranges for which the alerts did not change, the odds of medication change generally increased or stayed the same. There was little evidence of medication de-intensification in any group. CONCLUSIONS: These findings suggest that the change in therapeutic targets was associated with a reduction in medication intensification among older adults with diabetes.

Accuracy-Based Vitamin D Survey: Six Years of Quality Improvement Guided by Proficiency Testing.

CONTEXT.­: The goal of the College of American Pathologists Accuracy-Based Proficiency Testing Program is to promote the quality, standardization, and harmonization of clinical laboratory results through proficiency testing specimens that are free from matrix effects, have target values that are traceable to reference methods, and that probe the limitations of current methods. OBJECTIVE.­: To summarize the first 6 years of the Accuracy-Based Vitamin D Survey and highlight key insights from the data generated as it relates to assay performance. DESIGN.­: Accuracy-based challenges were created by using pooled human serum samples. Certain samples were derived from participants in an institutional review board-approved protocol in which vitamin D-deficient participants were treated with ergocalciferol (vitamin D2). Reference targets for the survey were set by the Centers for Disease Control and Prevention using isotope-dilution liquid chromatography-tandem mass spectrometry. Each method was compared with the reference method procedure over the course of the program (n = 43 proficiency testing samples). RESULTS.­: Linear regression versus the reference method procedure revealed proportional biases across the methods, ranging from 0.0% to 16.7%. Pearson correlation coefficients (r2) ranged from 0.902 to 0.996. Results were influenced by the concentration of 25-hydroxyvitamin D2 as well as the C-3 epimer of 25-hydroxyvitamin D3. During the 6 years, 2 manufacturers altered their assays to match the reference method procedure more closely. CONCLUSIONS.­: There is considerable bias, both proportional bias and sample-specific matrix effects, affecting many assays. This ongoing accuracy-based proficiency testing program for vitamin D will provide the data needed for laboratories and manufacturers to improve their assays and thereby patient care.

Improving Care in Older Patients with Diabetes: A Focus on Glycemic Control.

Diabetes affects more than 25% of Americans older than age 65 years. The medical care of older patients must differ from the care of their younger counterparts. Older patients are at high risk of drug toxicity. A hemoglobin A1c (HbA1c) level less than 7.0% has historically been the goal of all patients with diabetes, regardless of age. Recent research has demonstrated that using medications to achieve such tight glycemic control is not necessary and is often not safe.This article discusses the seminal research findings that strongly suggest that HbA1c goals should be relaxed in older patients. The authors then recommend an age-specific and functionally appropriate HbA1c reference range for patients receiving medications to improve glycemic control. Other interventions are suggested that should make diabetes care safer in older patients receiving hypoglycemic medications.

Trends in Prostate-specific Antigen Screening, Prostate Biopsies, Urology Visits, and Prostate Cancer Treatments From 2000 to 2012.

OBJECTIVE: To determine whether the rates of prostate-specific antigen (PSA) screening, related biopsies and subsequent prostate cancer utilization decreased between 2000 and 2012 in a large, managed care organization. METHODS: Male members of Kaiser Permanente Southern California who were aged ≥40 years and had no history of prostate cancer (N = 15,326) were passively followed through electronic health plan files from January 1, 2000, through December 31, 2012 (N = 1,539,469). The rates of PSA testing, elevated PSA tests, prostate biopsies, prostate cancer treatment (surgery and radiation), and urology visits were calculated per year among eligible men and stratified by age group. RESULTS: A 59% decrease in PSA screening occurred among men aged ≥75 years beginning in 2008, followed by 49% in ages 65-74, 20% in ages 50-64, and 33% in ages 40-49 years in 2009. However, the number of elevated PSA tests remained largely unchanged in all groups except in men aged ≥75 years (45% decrease). Prostate biopsy rates and urology visits remained consistent among elderly men. CONCLUSION: Among men in this managed care setting, although there was a sharp decline in PSA testing among men aged ≥75 years after 2008, prostate biopsy rates remained constant, and subsequent prostate cancer treatment remained highest among men in this age group. These results suggest that the guidelines recommending against PSA and the subsequent provider-targeted interventions implemented in this system resulted in decreased screening across age groups and potentially led to more discriminant screening among those aged ≥75 years.

Multilectin affinity chromatography for characterization of multiple glycoprotein biomarker candidates in serum from breast cancer patients.

BACKGROUND: Glycoproteins are often associated with cancer and are important in serum studies, for which glycosylation is a common posttranslational modification. METHODS: We used multilectin affinity chromatography (M-LAC) to isolate glycoproteins from the sera of breast cancer patients and controls. The proteins were identified by HPLC-tandem mass spectrometry (MS/MS) analysis of the corresponding tryptic digests. We used the FuncAssociate Gene Ontology program for association analysis of the identified proteins. Biomarker candidates in these groups were comparatively quantitated by use of peak area measurements, with inclusion of an internal standard. We analyzed data for concordance within the ontology association groups for vector of change with the development of breast cancer. RESULTS: Detection of the known low-concentration biomarker HER-2 (8-24 mug/L) enabled us to establish a dynamic range of 10(6), relative to the amount of albumin, for the depletion step. We then used ELISA to confirm this range. Proteins associated with lipid transport and metabolism, cell growth and maintenance, ion homeostasis, and protease inhibition were found to be differentially regulated in serum from women with breast cancer compared with serum from women without breast cancer. CONCLUSIONS: M-LAC for isolation of the serum glycoproteome, coupled with liquid chromatography-MS/MS and the use of gene ontology associations, can be used to characterize large panels of candidate markers, which can then be evaluated in a particular patient population.

Efficient method for the proteomic analysis of fixed and embedded tissues.

Formalin-fixed and paraffin-embedded (FFPE) tissues present a particular challenge for proteomic analysis. Yet, most of the archived tissues in hospitals and tissue banks worldwide are only available in this form. We have developed conditions for removal of the embedding medium and protein digestion, such that informative tryptic peptides are released from fixed proteins which are suitable for analysis by liquid chromatography-mass spectrometry (LC-MS). We demonstrate that the peptide identifications made by this approach compare favorably to those made from matched fresh frozen tissue. Moreover, we demonstrate that a high level of sequence coverage can be observed for proteins of interest.

Comparison of pooled fresh frozen serum to proficiency testing material in College of American Pathologists surveys: cortisol and immunoglobulin E.

CONTEXT: The College of American Pathologists (CAP) provides proficiency testing (PT) surveys to laboratories around the world. OBJECTIVES: To compare diagnostic assay methods for serum/plasma cortisol and immunoglobulin (Ig) E in terms of their bias and precision, to determine how well CAP PT specimens simulate human serum, and to reassess proficiency test grading criteria in light of these findings. DESIGN: A participant-blinded, prospective trial. One vial of pooled fresh frozen serum (FFS) and 4 different admixtures of PT material (PTM) were sent to laboratories participating in PT surveys. PARTICIPANTS: Laboratories providing cortisol (>1000) or IgE (>230) results among the subscribers to the CAP surveys, Ligand (General) 2003, set K/KN-A and Chemistry 2003, set C-C. MAIN OUTCOME MEASURES: The main outcome measures were (1) bias among laboratories using the same method (peer groups), defined relative to the median of method means (MedMM); (2) imprecision as measured by the SD and coefficient of variation (CV) about each method mean; and (3) total error across laboratories for the FFS cortisol results, defined as |Bias Relative to Reference Method| + 2 SD. RESULTS: Cortisol method biases, relative to MedMM, ranged from -22% to 9% for the FFS challenge and from -24% to 36% for comparable PTM challenges. The method biases, relative to the reference method, ranged from -3% to 19% for the FFS challenge. The cortisol method CVs ranged from 4.2% to 13.6% for the FFS challenge and from 4.7% to 12.7% for comparable PTM challenges. Total error across laboratories ranged from 1.4 to 6.9 microg/dL (39 to 190 nmol/L) for the FFS challenge. Immunoglobulin E method biases, relative to MedMM, ranged from -8% to 9% for the FFS challenge and from -7% to 5% for comparable PTM challenges. The IgE method CVs ranged from 3.6% to 6.7% for the FFS challenge and from 3.4% to 9.8% for comparable PTM challenges. CONCLUSIONS: The bias for cortisol results was less with FFS than with PTM, but imprecision was comparable. The FFS MedMM was 8.5% higher than the reference value. Fresh frozen serum and PTM bias and imprecision for IgE methods were each less than 10%. Because some of the methods demonstrated greater bias when analyzing PTM than FFS, peer group grading of both these analytes is appropriate.

Total long-term within-laboratory precision of cortisol, ferritin, thyroxine, free thyroxine, and thyroid-stimulating hormone assays based on a College of American Pathologists fresh frozen serum study: do available methods meet medical needs for precision?

CONTEXT: It is important that the total long-term precision of laboratory methods meet the medical needs of the patients being served. OBJECTIVES: To determine the long-term within- and between-laboratory variation of cortisol, ferritin, thyroxine, free thyroxine, and thyroid-stimulating hormone measurements using commonly available methods and to determine if these variations are within accepted medical needs. DESIGN: Two vials of pooled frozen serum were mailed 6 months apart to laboratories participating in 2 separate College of American Pathologists surveys. The data from those laboratories that analyzed an analyte in both surveys were used to determine for each method the total variance and the within- and between-laboratory components. SETTING: The study included the A mailing of the 2003 College of American Pathologists Ligand Survey and the C mailing of the Chemistry Survey. MAIN OUTCOME MEASURES: For each analyte, total variance was partitioned into within- and between-laboratory components for each analytic method. The within-laboratory variations were then compared with imprecision criteria based on biological variation. PARTICIPANTS: The laboratories that reported results on the same analyte using the same method in both surveys. RESULTS: For each analyte, the median of the long-term within-laboratory variances of each peer group was 78% to 95% of its total-survey variance, and the median long-term within-laboratory coefficients of variation varied from 5.1% to 7.6%. The number of methods that met within-laboratory imprecision goals based on biological criteria were 5 of 5 for cortisol; 5 of 7 for ferritin; 0 of 7 for thyroxine and free thyroxine; and 8 of 8 for thyroid-stimulating hormone. CONCLUSIONS: For all analytes tested, the total within-laboratory component of variance was the major source of variability in this study. In addition, there are several methods, especially for thyroxine and free thyroxine, that may not meet analytic goals in terms of their imprecision.

Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3).

The requirement for prefractionation in proteomic analysis is linked to the challenge of performing such an analysis on complex biological samples and identifying low level components in the presence of numerous abundant housekeeping and structural proteins. The employment of a preliminary fractionation step results in a reduction of complexity in an individual fraction and permits more complete liquid chromatography/mass spectrometry (LC/MS) analysis. Free flow electrophoresis (FFE), a solution-based preparative isoelectric focusing technique, fractionates and enriches protein fractions according to their charge differences and is orthogonal in selectivity to the popular reversed phase high performance liquid chromatography (HPLC) fractionation step. In this paper, we explored the advantages of a combination of FFE and liquid chromatography/mass spectrometry to extend the dynamic range of a proteomic analysis of a complex cell lysate. In this study, the whole cell lysate of a chronic myelogeneous leukemia cell line, K562/CR3, was prefractionated by FFE into 96 fractions spanning pH 3-12. Of these, 35 fractions were digested with trypsin and then analyzed by LC/MS. Depending on the algorithm used for peptide assignment from MS/MS data, at least 319 proteins were identified through database searches. The results also suggested that pI could serve as an additional criterion besides peptide fragmentation pattern for protein identification, although in some cases, a pI shift might indicate post-translational modification. In summary, this study demonstrated that free flow electrophoresis provided a useful prefractionation step for proteomic analysis and when combined with LC/MS allowed the identification of significant number of low level proteins in complex samples.

Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling.

The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., approximately 10 000 cells, which typically contain roughly 1-4 microg protein. The 16O/18O labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and 16O/18O labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the 16O/18O post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 microg. Next, protein expression was compared between 10 000 cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then 16O/18O labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate dehydrogenase, actin and 14-3-3 protein xi/delta were found to be significantly up-regulated in the breast tumor cells.

Placental villitis of unclear etiology during ovum donor in vitro fertilization pregnancy.

OBJECTIVE: Preliminary observations by a single pathologist at our institution revealed a 75% incidence of villitis of unexplained etiology in ovum donor in vitro fertilization pregnancies. Because the incidence of villitis of unexplained etiology in the general population is approximately 10%, we conducted a controlled study to compare the incidence of villitis of unexplained etiology in ovum donor in vitro fertilization pregnancies to that in in vitro fertilization pregnancies that do not use donated ova. STUDY DESIGN: Placental specimens of ovum donor in vitro fertilization pregnancies were matched randomly with pregnancies that resulted from both fresh and frozen/thawed native oocyte in vitro fertilization from March 5, 1995, to October 10, 2001, and examined in a blinded fashion by a single pathologist (D. J. R.) for villitis of unexplained cause. The incidence of villitis of unexplained etiology was analyzed in 27 patients who underwent ovum donor in vitro fertilization versus 37 patients who underwent native oocyte in vitro fertilization. RESULTS: Villitis of unexplained cause occurred in 22.2% of ovum donor in vitro fertilization pregnancies, 10.8% of native oocyte in vitro fertilization pregnancies (fresh and frozen/thawed combined), and 14.3% of frozen/thawed cycles (P=.21). CONCLUSION: Although the incidence was not statistically different than in in vitro fertilization that used native maternal oocytes, there was a 2-fold increase in villitis of unexplained cause in the ovum donor in vitro fertilization placentas, which suggests that immune-related disorders may be increased in ovum donor pregnancies.

Laser capture microdissection and advanced molecular analysis of human breast cancer.

Advances in comprehensive genomic and proteomic technologies are providing researchers with an unprecedented opportunity for high-throughput molecular analysis of human breast cancer. Adaptation of these technologies to laser capture microdissection (LCM) is poised to exert dramatic change on the pace of breast cancer research. Although technical limitations have impeded the coupling of these high-throughput technologies to LCM, recent advances have allowed for the successful application of this cellular-based approach to breast cancer, and the results of such studies have provided researchers with unique insight into the disease. This approach holds great potential for rapid advancement in our understanding of breast cancer, and it is hoped that such advancements will lead to novel predictive and therapeutic strategies for women with the disease. This review outlines the current status of the adaptation of advanced molecular technologies to LCM and highlights recent studies in which this approach has been applied to human breast cancer.