Inadequate Reporting of Analytical Characteristics of Biomarkers Used in Clinical Research: A Threat to Interpretation and Replication of Study Findings.

BACKGROUND: Analytical characteristics of methods to measure biomarkers determine how well the methods measure what they claim to measure. Transparent reporting of analytical characteristics allows readers to assess the validity and generalizability of clinical studies in which biomarkers are used. Our aims were to assess the reporting of analytical characteristics of biomarkers used in clinical research and to evaluate the extent of reported characterization procedures for assay precision. METHODS: We searched 5 medical journals (Annals of Internal Medicine, JAMA: The Journal of the American Medical Association, The Lancet, The New England Journal of Medicine, and PLOS Medicine) over a 10-year period for the term "biomarker" in the full-text field. We included studies in which biomarkers were used for inclusion/exclusion of study participants, for patient classification, or as a study outcome. We tabulated the frequencies of reporting of 11 key analytical characteristics (such as analytical accuracy of test results) in the included studies. RESULTS: A total of 544 studies and 1299 biomarker uses met the inclusion criteria. No information on analytical characteristics was reported for 67% of the biomarkers. For 65 biomarkers (3%), ≥4 characteristics were reported (range, 4-8). The manufacturer of the measurement procedure could not be determined for 688 (53%) of the 1299 biomarkers. The extent of assessments of assay imprecision, when reported, did not meet expectations for clinical use of biomarkers. CONCLUSIONS: Reporting of the analytical performance of biomarker measurements is variable and often absent from published clinical studies. We suggest that readers need fuller reporting of analytical characteristics to interpret study results, assess generalizability of conclusions, and compare results among clinical studies.

Therapeutic plasma exchange as a therapeutic modality for the treatment of IVIG complications.

Intravenous immunoglobulin (IVIG) is used for the treatment of a number of inflammatory conditions. Hemolysis due to passive transfer of blood group antibodies is a well recognized complication of IVIG therapy. Therapy is largely supportive and consists of blood product support and hemodialysis. We report the use of therapeutic plasma exchange (TPE) as adjunct therapy for three patients with complications attributed to IVIG. Two patients had hemolysis attributed to IVIG; one patient was blood group A and the other blood group O. The third patient was an orthotopic heart transplant recipient with a type A donor heart, and anti-A antibodies detected after infusion of IVIG for suspected antibody mediated rejection. Two patients had anti-A titers available that decreased after initiation of plasma exchange. The blood group O patient with hemolysis had a gradual stabilization of hemoglobin and resolution of the positive DAT. TPE may be useful therapy for patients with severe hemolysis caused by IVIG or at risk for tissue damage by blood group antibodies.

How do we manage cardiopulmonary bypass coagulopathy?

BACKGROUND: Patients who undergo cardiopulmonary bypass (CPB) are at risk for coagulopathy. Suboptimal turnaround time (TAT) of laboratory coagulation testing results in empiric administration of blood products to treat massive bleeding. We describe our initiative in establishing the coagulation-based hemotherapy (CBH) service, a clinical pathology consultation service that uses rapid TAT coagulation testing and provides comprehensive assessment of bleeding in patients undergoing CPB. A transfusion algorithm that treats the underlying cause of coagulopathy was developed. STUDY DESIGN AND METHODS: The coagulation testing menu includes all aspects of coagulopathy with close proximity of the laboratory to the operating room to allow for rapid test results. The hemotherapy pathologist monitors laboratory results at several stages in surgery and uses a comprehensive algorithm to monitor a patient's hemostasis. The optimal number and type of blood products are selected when the patient is taken off CPB. RESULTS: The CBH service was consulted for 44 ventricular assist device implants, 30 heart transplants, and 31 other cardiovascular surgeries from May 2012 through November 2013. The TAT for laboratory tests was 15 minutes for complete blood count, antithrombin, and coagulation panel and 30 minutes for VerifyNow and thromboelastography, in comparison to 45 to 60 minutes in normal settings. The transfusion algorithms were used with optimal administration of blood components with preliminary data suggestive of reduced blood product usage and better patient outcomes. CONCLUSION: We described the successful introduction of a novel pathology consultation service that uses a rapid TAT coagulation testing menu with transfusion algorithms for improved management of CPB patients.

Bidirectional (negative/positive) interference of oleandrin and oleander extract on a relatively new Loci digoxin assay using Vista 1500 analyzer.

BACKGROUND: Oleander interferes with serum digoxin measurements using various immunoassays. The potential interference of oleander and its active ingredient, oleandrin, with a relatively new homogenous sequential chemiluminescent digoxin assay based on luminescent oxygen channeling technology (LOCI digoxin assay, Siemens Diagnostics) has not been previously reported. METHODS: Aliquots of a digoxin-free serum pool were supplemented with increasing concentrations of oleandrin, or with oleander extract, followed by measuring the apparent digoxin concentrations using the LOCI digoxin assay using Vista 1500 analyzer. Mice were fed oleandrin or oleander extract, and their blood digoxin levels at 1 and 2 h were measured with the LOCI digoxin assay. In addition, two digoxin serum pools were prepared by combining sera of patients receiving digoxin; aliquots of both pools were supplemented with oleandrin or oleander extract and digoxin concentrations were again measured. Attempts to overcome this interference were made by measuring free digoxin concentration using a third digoxin pool. RESULTS: Significant apparent digoxin concentrations were observed after supplementing aliquots of the drug-free serum pool with oleandrin or oleander extract. Mice fed with oleandrin or oleander extract also showed apparent digoxin levels 1 and 2 h after feeding. Digoxin values were also falsely lower or elevated (bidirectional interference) when aliquots of digoxin serum pools were further supplemented with oleandrin or oleander extract depending on concentration; this interference was not eliminated by free digoxin monitoring. CONCLUSIONS: Oleandrin interferes with LOCI digoxin assay.

Complement factor C7 contributes to lung immunopathology caused by Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) remains a significant global health burden despite the availability of antimicrobial chemotherapy. Increasing evidence indicates a critical role of the complement system in the development of host protection against the bacillus, but few studies have specifically explored the function of the terminal complement factors. Mice deficient in complement C7 and wild-type C57BL/6 mice were aerosol challenged with MTB Erdman and assessed for bacterial burden, histopathology, and lung cytokine responses at days 30 and 60 post-infection. Macrophages isolated from C7 -/- and wild-type mice were evaluated for MTB proliferation and cytokine production. C7 -/- mice had significantly less liver colony forming units (CFUs) at day 30; no differences were noted in lung CFUs. The C7 deficient mice had markedly reduced lung occlusion with significantly increased total lymphocytes, decreased macrophages, and increased numbers of CD4+ cells 60 days post-infection. Expression of lung IFN-γ and TNF-α was increased at day 60 compared to wild-type mice. There were no differences in MTB-proliferation in macrophages isolated from wild-type and knock-out mice. These results indicate a role for complement C7 in the development of MTB induced immunopathology which warrants further investigation.

Determination of vancomycin and daptomycin MICs by different testing methods for methicillin-resistant Staphylococcus aureus.

Vancomycin and daptomycin MICs from 161 isolates of methicillin-resistant Staphylococcus aureus (MRSA) were compared using commercial and in-house broth microdilution, Etest, and common automated methods. Vancomycin Etest MICs were higher than those of other methods, whereas the MICs for daptomycin testing were comparable. Vancomycin MICs vary depending on the testing methodology.

Predictors of relapse of methicillin-resistant Staphylococcus aureus bacteremia after treatment with vancomycin.

The risk factors for relapse of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia after vancomycin treatment are unknown. Diversilab typing was used to classify recurrent bacteremia as relapse or reinfection. Bacteremia for >7 days and staphylococcal cassette chromosome mec element (SCCmec) type II were independently associated with relapse of MRSA bacteremia after vancomycin treatment.

IL-6 mediates 11ßHSD type 2 to effect progression of the mycobacterial cord factor trehalose 6,6'-dimycolate-induced granulomatous response.

Granulomatous structures are highly dynamic during active mycobacterial infection, with accompanying responsive inflammation contributing to modulation of pathology throughout the course of disease. The heightened inflammatory response coinciding with initiation and maintenance of newly developing granulomatous structures must be limited to avoid excessive damage to bystander tissue. Modulating the cellular bioavailability of glucocorticoids by local regulation of 11ßHSD enzymes within responding tissue and parenchyma would allow controlled inflammatory response during infection. Mycobacterial glycolipid trehalose 6,6'-dimycolate was used to induce strong pulmonary granulomatous inflammation immunopathology. Pulmonary corticosterone was significantly increased at days 3 and 5 after administration. An inverse relationship of 11ßHSD1 and 11ßHSD2 message correlated with pathology development. Immunohistochemical analysis also demonstrated that 11ßHSD2 is expressed in proximity to granulomatous lesions. A role for pro-inflammatory IL-6 cytokine in regulation of converting enzymes to control the granulomatous response was confirmed using gene-disrupted IL-6-/- mice. A model is proposed linking IL-6 to endocrine-derived factors which allows modification of active corticosterone into inert 11-dehydrocorticosterone at the site of granuloma formation to limit excessive parenchymal damage.

Immunopathology of postprimary tuberculosis: increased T-regulatory cells and DEC-205-positive foamy macrophages in cavitary lesions.

Postprimary tuberculosis occurs in immunocompetent people infected with Mycobacterium tuberculosis. It is restricted to the lung and accounts for 80% of cases and nearly 100% of transmission. Little is known about the immunopathology of postprimary tuberculosis due to limited availability of specimens. Tissues from 30 autopsy cases of pulmonary tuberculosis were located. Sections of characteristic lesions of caseating granulomas, lipid pneumonia, and cavitary stages of postprimary disease were selected for immunohistochemical studies of macrophages, lymphocytes, endothelial cells, and mycobacterial antigens. A higher percentage of cells in lipid pneumonia (36.1%) and cavitary lesions (27.8%) were positive for the dendritic cell marker DEC-205, compared to granulomas (9.0%, P < .05). Cavities contained significantly more T-regulatory cells (14.8%) than found in lipid pneumonia (5.2%) or granulomas (4.8%). Distribution of the immune cell types may contribute to the inability of the immune system to eradicate tuberculosis.

Thrombocytopenia including immune thrombocytopenia after receipt of mRNA COVID-19 vaccines reported to the Vaccine Adverse Event Reporting System (VAERS).

BACKGROUND: The objective of this study is to assess cases of thrombocytopenia, including immune thrombocytopenia (ITP), reported to the Vaccine Adverse Event Reporting System (VAERS) following vaccination with mRNA COVID-19 vaccines. METHODS: This case-series study analyzed VAERS reports of thrombocytopenia after vaccination with Pfizer-BioNTech COVID-19 Vaccine or Moderna COVID-19 Vaccine. RESULTS: Fifteen cases of thrombocytopenia were identified among 18,841,309 doses of Pfizer-BioNTech COVID-19 Vaccine and 13 cases among 16,260,102 doses of Moderna COVID-19 Vaccine. The reporting rate of thrombocytopenia was 0.80 per million doses for both vaccines. Based on an annual incidence rate of 3.3 ITP cases per 100,000 adults, the observed number of all thrombocytopenia cases, which includes ITP, following administration of mRNA COVID-19 vaccines is not greater than the number of ITP cases expected. CONCLUSIONS: The number of thrombocytopenia cases reported to VAERS does not suggest a safety concern attributable to mRNA COVID-19 vaccines at this time.

Myopericarditis after vaccination, Vaccine Adverse Event Reporting System (VAERS), 1990-2018.

BACKGROUND: Myopericarditis after vaccination has been sporadically reported in the medical literature. Here, we present a thorough descriptive analysis of reports to a national passive vaccine safety surveillance system (VAERS) of myopericarditis after vaccines licensed for use in the United States. METHODS: We identified U.S. reports of myopericarditis received by VAERS during 1990-2018 that met a published case definition for myopericarditis or were physician-diagnosed. We stratified analysis by age group (<19, 19-49, ≥50 years), describing reports by serious/non-serious status, sex, time to symptom onset after vaccination, vaccine(s) administered, and exposure to other known causes of myopericarditis. We used Empirical Bayesian data mining to detect disproportionate reporting of myopericarditis after vaccination. RESULTS: VAERS received 620,195 reports during 1990-2018: 708 (0.1%) met the case definition or were physician-diagnosed as myopericarditis. Most (79%) myopericarditis reports described males; 69% were serious; 72% had symptom onset ≤ 2 weeks postvaccination. Overall, smallpox (59%) and anthrax (23%) vaccines were most commonly reported. By age, among persons aged < 19 years, Haemophilus influenzae type b (22, 22%) and hepatitis B (18, 18%); among persons aged 19-49 years smallpox (387, 79%); among persons aged ≥ 50 years inactivated influenza (31, 36%) and live attenuated zoster (19, 22%) vaccines were most commonly reported. The vaccines most commonly reported remained unchanged when excluding 138 reports describing other known causes of myopericarditis. Data mining revealed disproportionate reporting of myopericarditis only after smallpox vaccine. CONCLUSIONS: Despite the introduction of new vaccines over the years, myopericarditis remains rarely reported after vaccines licensed for use in the United States. In this analysis, myopericarditis was most commonly reported after smallpox vaccine, and less commonly after other vaccines.

Clinical characteristics, outcomes, and microbiologic features associated with methicillin-resistant Staphylococcus aureus bacteremia in pediatric patients treated with vancomycin.

Vancomycin is the first-line therapy for methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, but its efficacy in adult patients has been questioned. Less is known about the outcomes of MRSA bacteremia treated with vancomycin in pediatric patients. This study reviews the outcomes and clinical characteristics of MRSA bacteremia in children treated with vancomycin and characterizes the microbiologic and molecular features of the bloodstream isolates. A retrospective cohort study was conducted among pediatric patients with MRSA bacteremia treated with vancomycin for >5 days from 1 August 2005 to 31 May 2007 in a large tertiary care center. MRSA bloodstream isolates were characterized by antimicrobial susceptibility testing, PCR analysis of virulence genes, and Diversilab typing. Clinical records were reviewed for outcomes and comorbidities. A total of 22 pediatric patients with MRSA bacteremia were identified. Eleven cases (50.0%) were considered vancomycin treatment failures. Features significantly associated with vancomycin treatment failure were prematurity (P = 0.02) and isolates positive for Panton-Valentine leukocidin (PVL) (P = 0.008). Features typically associated with community-associated MRSA strains were identified in hospital-associated isolates. A dominant clone was not responsible for the high number of treatment failures. Further studies are needed to determine if vancomycin should be the first-line treatment for MRSA bacteremia in premature infants and for PVL-positive isolates.

Adaptive NK cells in people exposed to Plasmodium falciparum correlate with protection from malaria.

How antibodies naturally acquired during Plasmodium falciparum infection provide clinical immunity to blood-stage malaria is unclear. We studied the function of natural killer (NK) cells in people living in a malaria-endemic region of Mali. Multi-parameter flow cytometry revealed a high proportion of adaptive NK cells, which are defined by the loss of transcription factor PLZF and Fc receptor γ-chain. Adaptive NK cells dominated antibody-dependent cellular cytotoxicity responses, and their frequency within total NK cells correlated with lower parasitemia and resistance to malaria. P. falciparum-infected RBCs induced NK cell degranulation after addition of plasma from malaria-resistant individuals. Malaria-susceptible subjects with the largest increase in PLZF-negative NK cells during the transmission season had improved odds of resistance during the subsequent season. Thus, antibody-dependent lysis of P. falciparum-infected RBCs by NK cells may be a mechanism of acquired immunity to malaria. Consideration of antibody-dependent NK cell responses to P. falciparum antigens is therefore warranted in the design of malaria vaccines.

Human Papillomavirus and Its Testing Assays, Cervical Cancer Screening, and Vaccination.

Human papillomavirus (HPV) was found to be the causative agent for cervical cancer in the 1980s with almost 100% of cervical cancer cases testing positive for HPV. Since then, many studies have been conducted to elucidate the molecular basis of HPV, the mechanisms of carcinogenesis of the virus, and the risk factors for HPV infection. Traditionally, the Papanicolaou test was the primary screening method for cervical cancer. Because of the discovery and evolving understanding of the role of HPV in cervical dysplasia, HPV testing has been recommended as a new method for cervical cancer screening by major professional organizations including the American Cancer Society, American Society for Colposcopy and Cervical Pathology, and the American Society for Clinical Pathology. In order to detect HPV infections, many sensitive and specific HPV assays have been developed and used clinically. Different HPV assays with various principles have shown their unique advantages and limitations. In response to a clear causative relationship between high-risk HPV and cervical cancer, HPV vaccines have been developed which utilize virus-like particles to create an antibody response for the prevention of HPV infection. The vaccines have been shown in long-term follow-up studies to be effective for up to 8 years; however, how this may impact screening for vaccinated women remains uncertain. In this chapter, we will review the molecular basis of HPV, its pathogenesis, and the epidemiology of HPV infection and associated cervical cancer, discuss the methods of currently available HPV testing assays as well as recent guidelines for HPV screening, and introduce HPV vaccines as well as their impact on cervical cancer screening and treatments.

Assessment of thyroid function in intensive care unit patients by liquid chromatography tandem mass spectrometry methods.

OBJECTIVES: Patients with non-thyroidal illness syndrome have many abnormalities in thyroid hormone tests. Such patients have medical comorbidities associated with low serum proteins and are on multiple medications that interfere with thyroid hormone measurement by immunoassay platforms. It is unknown if these thyroid hormone measurements reflect physiologic conditions or if they are artifacts of testing methodology. METHODS: Fifty patients were selected from the intensive care unit (ICU) from our institution. Total and free thyroid hormones in plasma were measured by gold standard liquid chromatography-tandem mass spectrometry (LC-MSMS). The results were compared to the Roche Cobas 6000. Patient medical comorbidities and binding protein levels were assessed. RESULTS: Concentrations of total 3,5,5'-triidothyronine (TT3) and total thyroxine (TT4) were significantly more likely to be low by LC-MSMS compared to immunoassay. Free 3,5,5'-triidothyronine (FT3) levels were similar by immunoassay and LC-MSMS. However, FT4 concentrations were mildly elevated for many patients when measured by ultrafiltration LC-MSMS (19/50, 38%) compared to 1/50 (2%) when measured by immunoassay (p=0.0001). Decreased albumin and thyroxine binding globulin were common and patients were on an average of 11.7±5.0 medications, all factors known to interfere with results found on immunoassays. CONCLUSIONS: Marked discrepancies in thyroid hormone measurement were noted between reference LC-MSMS and a common immunoassay platform. It is hypothesized that T4 binding to low affinity albumin is displaced by several drugs, raising concentrations of FT4 by LC-MSMS compared to immunoassay, and that the immunoassay values are falsely decreased due to low binding proteins in our patient population.

Pathology Consultation on Patients With a Large Rh Immune Globulin Dose Requirement.

OBJECTIVES: To review the differential diagnosis and laboratory issues for women with a large calculated dose of Rh immune globulin (RhIG). METHODS: A case-based approach is used to review the differential diagnosis of patients with a large calculated dose of RhIG, RhIG dosing for women with baseline elevations in hemoglobin F, the formulations of RhIG, and issues for the transfusion medicine service with the release of large doses of RhIG. RESULTS: A large fetomaternal bleed after delivery requiring multiple doses of RhIG is rare. Such patients may require intravenous RhIG to avoid multiple injections. Patients with a large percentage of circulating fetal RBCs should be evaluated for a disorder of hemoglobin synthesis and, if present, should have quantification of the circulating fetal RBCs by flow cytometry. CONCLUSIONS: Accurate laboratory evaluation of women with large fetomaternal bleeds is essential for appropriate RhIG administration.

DIAGNOSIS OF ENDOCRINE DISEASE: How reliable are free thyroid and total T3 hormone assays?

Hypothyroidism is a very common disorder worldwide, for which the usual treatment is monotherapy with levothyroxine (L-T4). However, a number of patients treated with L-T4 continue to report symptoms of hypothyroidism despite seemingly normal levels of thyroid-stimulating hormone (TSH), free-T3 (FT3) and free-T4 (FT4) measured by immunoassay. This review summarizes the limitations of the immunoassays commonly used to measure thyroid hormone levels and emphasizes the advantages of the role of liquid chromatography-tandem mass spectrometry (LC-MS/MS). Immunoassays for free thyroid hormone are affected by alterations in serum binding proteins that occur in many physiological and disease states. Multiple studies show falsely normal values for T3, FT3 and FT4 by immunoassay that are below the reference interval when measured by (ultrafiltration) LC-MS/MS, a reference method. We suggest evaluation of thyroid hormone levels by ultrafiltration LC-MS/MS for patients who continue to experience hypothyroid symptoms on LT-4. This may help identify the approximately 20% subset of patients who would benefit from addition of T3 to their treatment regimen (combination therapy).

Sirt1-Positive Lymphocytes in Acute Cellular Cardiac Allograft Rejection: Contributor to Pathogenesis and a Therapeutic Target.

Cardiac allograft rejection remains a problem, despite advances with immunosuppressants. Understanding the mechanisms behind rejection is essential for developing targeted therapies. The goal of this investigation is to explore Sirtuin 1 (Sirt1) as a therapeutic target for cardiac allograft rejection. Thirteen endomyocardial biopsy specimens with acute cellular rejection (grade 2R or 3R) were selected. CD3, CD4, CD8, CD20, CD68, T-cell intracytoplasmic antigen (TIA-1), and Sirt1 expressions were determined by immunohistochemical stains. Comparison of Sirt1 expression was made with 10 cases of grade 0R and grade 1R. Quantitative image analysis was performed. There were 2 cases of grade 3R and 11 cases of grade 2R acute cellular rejection. Sirtuin 1 expression was present in the majority of mononuclear cells (median percentage, 73.5; interquartile range, 51.2-100%); staining was also observed in cardiomyocytes. Twelve of the 13 cases (92.3%) had an elevated CD8/FoxP3 ratio, coinciding with acute cellular rejection. Sirtuin 1 expression in the nuclei of FoxP3+ cells can lead to deacetylation and inactivation of FoxP3 rendering the T-suppressor cells inactive and promoting acute cellular rejection. The use of a Sirt1 inhibitor may be a therapeutic option in expanding the functionality of the FoxP3+ T-suppressor cells and moderating the severity of such rejection.

Algorithmic Approach With Clinical Pathology Consultation Improves Access to Specialty Care for Patients With Systemic Lupus Erythematosus.

OBJECTIVES: Harris Health System (HHS) is a safety net system providing health care to the underserved of Harris County, Texas. There was a 6-month waiting period for a rheumatologist consult for patients with suspected systemic lupus erythematosus (SLE). The objective of the intervention was to improve access to specialty care. METHODS: An algorithmic approach to testing for SLE was implemented initially through the HHS referral center. The algorithm was further offered as a "one-click" order for physicians, with automated reflex testing, interpretation, and case triaging by clinical pathology. RESULTS: Data review revealed that prior to the intervention, 80% of patients did not have complete laboratory workups available at the first rheumatology visit. Implementation of algorithmic testing and triaging of referrals by pathologists resulted in decreasing the waiting time for a rheumatologist by 50%. CONCLUSIONS: Clinical pathology intervention and case triaging can improve access to care in a county health care system.