Developing a Prospective Gestational Lyme Disease Study.

Lyme disease in pregnancy is understudied. The few available reports of Borrelia infection during pregnancy collecting clinical outcomes, with or without confirmed fetal infection both in utero and neonatal, are limited to case reports and small series. Population-based studies are not available. We propose a prospective study of Borrelia infection during pregnancy based in obstetrical practices in both endemic and nonendemic areas, with long term follow-up of pregnancy outcomes and development assessment of offspring infected or exposed to Borrelia in utero using current serological, microscopic, culture, and molecular techniques. In addition to detection of Borrelia burgdorferi sensu stricto, additional Borrelia species and other pathogens known to be transmitted by ticks will be tested. Serial biospecimens including maternal and cord blood, maternal peripheral blood mononuclear cells and urine, and, when clinically indicated, amniotic fluid, chorionic villi, intrauterine cord blood, will be collected with clinical data, imaging, and for infections treatment medications. Offspring will be followed until age 5 years with annual developmental assessments to assess pregnancy outcomes. The study will require parallel development of a biorepository with strategies for management, data security and data sharing. A public-private partnership will be required to support the study.

Establishing a Pregnancy Lyme Disease Biobank.

Availability of relevant biological samples supports both basic science research and patient-centered clinical studies. Establishing a biorepository faces challenges at multiple levels. These tasks include defining mission definition and scope; selection of subjects and sample types; recruitment strategies; timing of collection in the patient's journey; sample logistics and processing; determining what clinical data to collect; ensuring sample integrity on transport, processing, and storage; defining governance structures and oversight responsibilities; clarifying sample provenance and ownership; establishing procedures for sample and data access; selecting testing to be performed routinely versus upon request, and management of results; data security; funding sources; and regulatory compliance. Establishing and maintaining a biorepository therefore requires careful planning, diligent and sustained execution, technical and financial resources, stakeholder support, and flexible and resilient management to respond to changing environments and needs.

Highlights of the 33rd annual scientific meeting of the Association of Medical Laboratory Immunologists (AMLI).

The annual meeting of the Association of Medical Laboratory Immunologists (AMLI) was convened virtually over the month of August. Prior to the emergence of the COVID-19 pandemic, AMLI's scientific committee had chosen the following topics as the focus of its 2020 meeting: Histocompatibility Testing and Transplant Immunology; Secondary Immunodeficiency and Immunotherapy Monitoring; ANA Update; and Emerging Infectious Diseases and New Algorithms for Testing. Given the central role of the discipline in the evaluation of the host response to infection, it was apt to add a separate session on antibody testing for SARS-CoV-2 infections to the original program. The current report provides an overview of the subjects discussed in the course of this meeting.

Prevalence and significance of serum 14-3-3η in juvenile idiopathic arthritis.

BACKGROUND: Prompt diagnosis of juvenile idiopathic arthritis (JIA) is important to avoid long term complications. Elevated serum 14-3-3η levels improve the diagnostic sensitivity of rheumatoid factor (RF) and cyclic citrullinated peptide (CCP) antibody in adult rheumatoid arthritis (RA), and have been associated with more severe phenotype. We investigated the prevalence and clinical significance of serum 14-3-3η in different types of JIA. METHODS: JIA patients (n = 151) followed by the Pediatric Rheumatology Core at Children's Hospital of Los Angeles were categorized into 5 groups: polyarticular JIA RF+ (PJIA RF+; n = 39), PJIA RF- (n = 39), psoriatic arthritis (PsA; n = 19), enthesitis-related arthritis (ERA; n = 18), and oligoarticular JIA (OJIA [control group]; n = 36). RF, CCP antibody, and 14-3-3η were measured for all patients. 14-3-3η serum levels > 0.2 ng/mL were considered positive. Disease activity was assessed by the Juvenile Arthritis Disease Activity Score-71 (JADAS-71). RESULTS: Elevated 14-3-3η levels were detected in 34/151 (23%) patients, and across all groups tested. Most patients with 14-3-3η had titers ≥4 times above the cutoff value. The majority (22, 65%) of 14-3-3η-positive patients were also positive for RF or CCP antibodies, 16 (47%) were positive for all 3, and 12 (35%) were single-positive for 14-3-3η. The highest prevalence of 14-3-3η was in PJIA RF+ patients (49%), followed by OJIA (22%). Positivity for 14-3-3η was not significantly associated with disease activity or age at diagnosis. CONCLUSION: Serum 14-3-3η can be detected in all forms of JIA tested but appears to be most common in PJIA RF+. 14-3-3η does not appear to correlate with disease activity in JIA.

14-3-3η Protein as a Potential Biomarker in Juvenile Idiopathic Arthritis.

The 14-3-3η (eta) protein was evaluated as a biomarker in a cohort of patients with juvenile idiopathic arthritis (JIA), as well as disease- and healthy-controls, to determine its potential clinical utility. In this case-control study, levels of 14-3-3η protein were evaluated in archival specimens from patients with JIA, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), as well as healthy pediatric controls. Just over 200 patients were evaluated, using specimens banked between 1990 and 2011. Comparisons were made to complete blood cell count (CBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP) antibodies, and anti-nuclear antibody (ANA) positivity. 14-3-3η at levels 0.2 ng/mL or higher was considered positive. Fisher's exact tests, odds ratios, 95% confidence intervals, and p-values were reported. 14-3-3η positivity was seen in all included JIA subtypes. The rate of positivity was the highest in RF-positive (pos) polyarticular JIA. In the disease and healthy controls, lower rates of positivity were observed. The frequency of 14-3-3η positivity among RF-positive and RF-negative (neg) polyarticular JIA patients, especially at values ≥0.5 ng/mL (associated with poor outcomes in adults), was also highest. Several JIA patients with 14-3-3η positivity developed RF and anti-CCP positivity later in their disease. Significant levels of 14-3-3η can be found in approximately 30% of RF-pos and RF-neg patients with polyarticular JIA. This protein may represent a new biomarker for polyarticular JIA, particularly RF-neg polyarticular JIA.

The Development of New Diagnostic Tests for Neurologic Disorders in the Commercial Laboratory Environment.

Development of new diagnostic tests in a commercial laboratory for neurologic disorders is challenging. Development occurs in a highly regulated environment. Relevant research infrastructure may not be readily available in-house and may require outsourcing with additional management and costs. Clinically characterized specimens for validation of biomarkers for esoteric diseases may be difficult to acquire, and market size may be difficult to predict. More common diseases with heterogeneous subsets may require better clinical definition. Absence of guidelines may delay health provider acceptance of novel testing. Regulatory agency approval and categorization of tests affects validation requirements and impacts market acceptance and reimbursement.

The rapid Bethesda assay is equivalent to the standard Bethesda assay for detection of factor IX inhibitors in patients with severe haemophilia B.

INTRODUCTION: The time-dependent nature of factor VIII (FVIII) inhibitors is well described, and the standard FVIII Bethesda assay used to measure inhibitors incorporates a 2-hour incubation. Despite case reports and reviews describing the immediate-acting nature of factor IX (FIX) inhibitors, many coagulation laboratories continue to use a traditional prolonged incubation for FIX Bethesda assays. To our knowledge, a comprehensive evaluation of the FIX Bethesda assay without incubation has not been reported. AIM: The goal of this study was to evaluate the performance of a rapid FIX Bethesda (ie no incubation) compared with the standard Bethesda assay (2-hour incubation). METHODS: The analysis used a Bethesda assay configured for either immediate testing or a 2-hour incubation. Samples from 14 haemophilia B patients with inhibitors and 9 non-human controls were tested. RESULTS: The two assays yielded similar performance overall. The average per cent difference in inhibitor titre between the rapid and standard FIX Bethesda assay was -3% (range -15% to +13%; P = .175) for patient samples and -2% (range -17% to +14%; P = .376) for controls. CONCLUSION: The rapid Bethesda assay showed good agreement with the standard Bethesda assay for determination of inhibitor levels in patients with severe haemophilia B. The rapid assay allows for faster assessment of inhibitors in patients with severe haemophilia B and has the potential to improve the ability of the coagulation laboratory to perform testing from a logistical viewpoint. Further studies involving larger numbers of patients would be important to confirm our findings.

Antinuclear Antibodies Testing Method Variability: A Survey of Participants in the College of American Pathologists' Proficiency Testing Program.

OBJECTIVE: This study was conducted to determine the spectrum of laboratory practices in antinuclear antibody (ANA) test target, performance, and result reporting. METHODS: A questionnaire on ANA testing was distributed by the Diagnostic Immunology and Flow Cytometry Committee of the College of American Pathologists (CAP) to laboratories participating in the 2016 CAP ANA proficiency survey. RESULTS: Of 5847 survey kits distributed, 1206 (21%) responded. ANA screening method varied: 55% indirect immunofluorescence assay, 21% ELISA, 12% multibead immunoassay, and 18% other methods. The name of the test indicated the method used in only 32% of laboratories; only 39% stated the method used on the report. Of 644 laboratories, 80% used HEp-2 cell substrate, 18% HEp-2000 (HEp-2 cell line engineered to overexpress SSA antigen, Ro60), and 2% other. Slides were prepared manually (67%) or on an automated platform (33%) and examined by direct microscopy (84%) or images captured by an automated platform (16%). Only 50% reported a positive result at the customary 1:40 dilution. Titer was reported to endpoint routinely by 43%, only upon request by 23%, or never by 35%. Of the laboratories, 8% did not report dual patterns. Of those reporting multiple patterns, 23% did not report a titer with each pattern. CONCLUSION: ANA methodology and practice, and test naming and reporting varies significantly between laboratories. Lack of uniformity in testing and reporting practice and lack of transparency in communicating the testing method may misdirect clinicians in their management of patients.

Reference intervals for lymphocyte subsets in preterm and term neonates without immune defects.

BACKGROUND: In 6.5 years of newborn screening for severe combined immunodeficiency in California, 3,252,156 infants had DNA from dried blood spots (DBSs) assayed for T-cell receptor excision circles. Infants with T-cell receptor excision circle values of less than a designated cutoff on a single DBS, 2 DBS samples with insufficient PCR amplification, or known genetic risk of immunodeficiency had peripheral blood complete blood counts and lymphocyte subsets assayed in a single flow cytometry laboratory. Cases in which immune defects were ruled out were available for analysis. OBJECTIVE: We sought to determine reference intervals for lymphocyte subsets in racially/ethnically diverse preterm and term newborns who proved to be unaffected by any T-lymphopenic immune disorder. METHODS: Effective gestational age (GA) was defined as GA at birth plus postnatal age at the time of sample collection. After determining exclusion criteria, we analyzed demographic and clinical information, complete and differential white blood cell counts, and lymphocyte subsets for 301 infants, with serial measurements for 33 infants. Lymphocyte subset measurements included total T cells, helper and cytotoxic T-cell subsets, naive and memory phenotype of each T-cell subset, B cells, and natural killer cells. RESULTS: Reference intervals were generated for absolute numbers and lymphocyte subsets from infants with effective GAs of 22 to 52 weeks. Sex and ethnicity were not significant determinants of lymphocyte subset counts in this population. Lymphocyte counts increased postnatally. CONCLUSION: This study provides a baseline for interpreting comprehensive lymphocyte data in preterm and term infants, aiding clinicians to determine which newborns require further evaluations for immunodeficiency.

Newborn Screening for Severe Combined Immunodeficiency and T-cell Lymphopenia in California, 2010-2017.

OBJECTIVES: Newborn screening for severe combined immunodeficiency (SCID) was instituted in California in 2010. In the ensuing 6.5 years, 3 252 156 infants in the state had DNA from dried blood spots assayed for T-cell receptor excision circles (TRECs). Abnormal TREC results were followed-up with liquid blood testing for T-cell abnormalities. We report the performance of the SCID screening program and the outcomes of infants who were identified. METHODS: Data that were reviewed and analyzed included demographics, nursery summaries, TREC and lymphocyte flow-cytometry values, and available follow-up, including clinical and genetic diagnoses, treatments, and outcomes. RESULTS: Infants with clinically significant T-cell lymphopenia (TCL) were successfully identified at a rate of 1 in 15 300 births. Of these, 50 cases of SCID, or 1 in 65 000 births (95% confidence interval 1 in 51 000-1 in 90 000) were found. Prompt treatment led to 94% survival. Infants with non-SCID TCL were also identified, diagnosed and managed, including 4 with complete DiGeorge syndrome who received thymus transplants. Although no cases of typical SCID are known to have been missed, 2 infants with delayed-onset leaky SCID had normal neonatal TREC screens but came to clinical attention at 7 and 23 months of age. CONCLUSIONS: Population-based TREC testing, although unable to detect immune defects in which T cells are present at birth, is effective for identifying SCID and clinically important TCL with high sensitivity and specificity. The experience in California supports the rapid, widespread adoption of SCID newborn screening.

Parvovirus B19V Nonstructural Protein NS1 Induces Double-Stranded Deoxyribonucleic Acid Autoantibodies and End-Organ Damage in Nonautoimmune Mice.

BACKGROUND: Viral infection is implicated in development of autoimmunity. Parvovirus B19 (B19V) nonstructural protein, NS1, a helicase, covalently modifies self double-stranded deoxyribonucleic acid (dsDNA) and induces apoptosis. This study tested whether resulting apoptotic bodies (ApoBods) containing virally modified dsDNA could induce autoimmunity in an animal model. METHODS: BALB/c mice were inoculated with (1) pristane-induced, (2) B19V NS1-induced, or (3) staurosporine-induced ApoBods. Serum was tested for dsDNA autoantibodies by Crithidia luciliae staining and enzyme-linked immunosorbent assay. Brain, heart, liver, and kidney pathology was examined. Deposition of self-antigens in glomeruli was examined by staining with antibodies to dsDNA, histones H1 and H4, and TATA-binding protein. RESULTS: The B19V NS1-induced ApoBod inoculation induced dsDNA autoantibodies in a dose-dependent fashion. Histopathological features of immune-mediated organ damage were evident in pristane-induced and NS1-induced ApoBod groups; severity scores were higher in these groups than in staurosporine-treated groups. Tissue damage was dependent on NS1-induced ApoBod dose. Nucleosomal antigens were deposited in target tissue from pristane-induced and NS1-induced ApoBod inoculated groups, but not in the staurosporine-induced ApoBod inoculated group. CONCLUSIONS: This study demonstrated proof of principle in an animal model that virally modified dsDNA in apoptotic bodies could break tolerance to self dsDNA and induce dsDNA autoantibodies and end-organ damage.

The role of the laboratory in the expanding field of neuroimmunology: Autoantibodies to neural targets.

Accelerated identification of autoantibodies associated with previously idiopathic neurological disease has provided insights into disease mechanisms, enhanced understanding of neurological function, and opportunities for improved therapeutic interventions. The role of the laboratory in the expanding field of neuroimmunology is critical as specific autoantibody identification provides guidance to clinicians in diagnosis, prognosis, tumor search strategies, and therapeutic interventions. The number of specific autoantibodies identified continues to increase and newer testing strategies increase efficiencies in the laboratory and availability to clinicians. The need for broadly targeted efficient testing is underscored by the variability in clinical presentation and tumor associations attributable to a specific autoantibody, and conversely the various autoantibody specificities that can be the cause of a given clinical presentation. While many of the antineural antibodies were first recognized in the setting of neoplastic disease, idiopathic autoimmune neurological disease in the absence of underlying tumor is increasingly recognized. Appropriation of therapeutic modalities used to treat autoimmune disease to treat these autoantibody mediated neurological diseases has improved patient outcomes. Interaction between clinicians and laboratorians is critical to our understanding of these diseases and optimization of the clinical benefits of our increasing knowledge in neuroimmunology.

Variability in Testing for Antineutrophil Cytoplasmic Antibodies: A Survey of Participants in the College of American Pathologists Proficiency Testing Program.

CONTEXT: -Variability in testing for antineutrophil cytoplasmic antibodies (ANCAs) contributes to confusion and controversy related to testing for vasculitis and other ANCA-associated diseases. OBJECTIVES: -To survey laboratory testing practices regarding ANCA testing and to investigate differences in testing algorithms. DESIGN: -Supplemental questions were sent to the 333 laboratories participating in the College of American Pathologists proficiency testing program for ANCA as part of the Special Immunology S2 Survey. RESULTS: -A total of 315 laboratories submitted responses to the supplemental questions. Only 88 of 315 participants (28%) reported using a combination of indirect immunofluorescence (IFA) and enzyme immunoassay (EIA) techniques as recommended by current guidelines, with a few additional labs using IFA and multiplex bead assay as an acceptable alternative to EIA. Other labs reported using only IFA, EIA, or multiplex bead assays. CONCLUSIONS: -A wide variety of testing algorithms are in use for ANCA testing despite evidence to suggest that a combination of IFA and EIA testing provides the most comprehensive information. Laboratories should inform clinicians clearly about testing practices and utility of testing in specific disease states.

An Internet-based technique for the identification of persons with symptoms of inflammatory polyarthritis of less than 12 weeks.

Identifying persons with early rheumatoid arthritis (RA) is a major challenge. The role of the Internet in making decisions about seeking care has not been studied. We developed a method for early diagnosis and referral using the Arthritis Foundation's website. A person with less than 3 months of joint pain symptom who has not yet sought medical attention was screened. Prescreened persons are linked to a self-scoring questionnaire and get a "likelihood" of RA statement. If "likely," the person is offered a free evaluation and biomarker testing performed by Quest Diagnostics. The system available only to Massachusetts's residents yielded a small steady flow of screen-positive individuals. Over 21 months, 43,244 persons took the Arthritis Foundation website prescreening questionnaire; 196 were from Massachusetts and 60 took the self-scoring algorithm. Of the 48 who screened positive, 29 set up an appointment for a free evaluation, but six never came in. Twenty-four subjects were evaluated and diagnosed independently by three rheumatologists. One met the 1987 American College of Rheumatology (ACR) criteria for RA and two met the 2010 ACR/EULAR RA criteria. The 24 examined individuals were contacted at a minimum of 1 year and asked to redo the case-finding questionnaire and asked about their health resource utilization during the interval. Seventeen of the 24 subjects responded, and 10 had seen a health professional. Three of the 17 had a diagnosis of RA; all were on at least methotrexate. Internet case finding was useful in identifying new potential RA cases. The system's performance characteristics are theoretically limited only by the number of study sites available. However, the major barrier may be that seeing a health professional is not a priority for many individuals with early symptoms.

Serum 14-3-3η is a novel marker that complements current serological measurements to enhance detection of patients with rheumatoid arthritis.

OBJECTIVE: Serum 14-3-3η is a novel joint-derived proinflammatory mediator implicated in the pathogenesis of rheumatoid arthritis (RA). In our study, we assessed the diagnostic utility of 14-3-3η and its association with standard clinical and serological measures. METHODS: A quantitative ELISA was used to assess 14-3-3η levels. Early (n=99) and established patients with RA (n=135) were compared to all controls (n=385), including healthy subjects (n=189). The sensitivity, specificity, positive and negative predictive values of 14-3-3η, and the likelihood ratios (LR) for RA were determined through receiver-operator curve analysis. The incremental value of adding 14-3-3η to anticitrullinated protein antibody (ACPA) and rheumatoid factor (RF) in diagnosing early and established RA was assessed. RESULTS: Serum 14-3-3η differentiated established patients with RA from healthy individuals and all controls (p<0.0001). A serum 14-3-3η cutoff of ≥0.19 ng/ml delivered a sensitivity and specificity of 77% and 93%, respectively, with corresponding LR positivity of 10.4. At this cutoff in early RA, 64% of patients with early RA were positive for 14-3-3η, with a corresponding specificity of 93% (LR+ of 8.6), while 59% and 57% were positive for ACPA or RF, respectively. When ACPA, RF, and 14-3-3η positivity were used in combination, 77 of the 99 patients (78%) with early RA were positive for any 1 of the 3 markers. Serum 14-3-3η did not correlate with C-reactive protein, erythrocyte sedimentation rate, or Disease Activity Score, but patients who were 14-3-3η-positive had significantly worse disease. CONCLUSION: Serum 14-3-3η is a novel RA mechanistic marker that is highly specific, associated with worse disease, and complements current markers, enabling a more accurate diagnosis of RA.

Newborn screening for severe combined immunodeficiency in 11 screening programs in the United States.

IMPORTANCE: Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100,000 births. OBJECTIVES: To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments. DESIGN: Epidemiological and retrospective observational study. SETTING: Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3,030,083 newborns screened with a TREC test. MAIN OUTCOMES AND MEASURES: Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked. RESULTS: Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58,000 infants (95% CI, 1/46,000-1/80,000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87% (45/52), 92% (45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia. CONCLUSIONS AND RELEVANCE: Newborn screening in 11 programs in the United States identified SCID in 1 in 58,000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined.