Detection of antinuclear antibodies: recommendations from EFLM, EASI and ICAP.

OBJECTIVES: Antinuclear antibodies (ANA) are important for the diagnosis of various autoimmune diseases. ANA are usually detected by indirect immunofluorescence assay (IFA) using HEp-2 cells (HEp-2 IFA). There are many variables influencing HEp-2 IFA results, such as subjective visual reading, serum screening dilution, substrate manufacturing, microscope components and conjugate. Newer developments on ANA testing that offer novel features adopted by some clinical laboratories include automated computer-assisted diagnosis (CAD) systems and solid phase assays (SPA). METHODS: A group of experts reviewed current literature and established recommendations on methodological aspects of ANA testing. This process was supported by a two round Delphi exercise. International expert groups that participated in this initiative included (i) the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group "Autoimmunity Testing"; (ii) the European Autoimmune Standardization Initiative (EASI); and (iii) the International Consensus on ANA Patterns (ICAP). RESULTS: In total, 35 recommendations/statements related to (i) ANA testing and reporting by HEp-2 IFA; (ii) HEp-2 IFA methodological aspects including substrate/conjugate selection and the application of CAD systems; (iii) quality assurance; (iv) HEp-2 IFA validation/verification approaches and (v) SPA were formulated. Globally, 95% of all submitted scores in the final Delphi round were above 6 (moderately agree, agree or strongly agree) and 85% above 7 (agree and strongly agree), indicating strong international support for the proposed recommendations. CONCLUSIONS: These recommendations are an important step to achieve high quality ANA testing.

Antibody Dependent Enhancement Due to Original Antigenic Sin and the Development of SARS.

Human coronavirus (HCoV) is one of the most common causes of respiratory tract infections throughout the world. Two phenomena observed so far in the development of the SARS-CoV-2 pandemic deserve further attention. First, the relative absence of clinical signs of infections in children, second, the early appearance of IgG in certain patients. From the point of view of immune system physiology, such an early rise of specific IgG is expected in secondary immune responses when memory to a cross-reactive antigen is present, usually from an earlier infection with a coronavirus. It is actually typical for the immune system to respond, to what it already knows, a phenomenon that has been observed in many infections with closely related viruses and has been termed "original antigenic sin." The question then arises whether such cross-reactive antibodies are protective or not against the new virus. The worst scenario would be when such cross-reactive memory antibodies to related coronaviruses would not only be non-protective but even enhance infection and the clinical course. Such a phenomenon of antibody dependent enhancement (ADE) has already been described in several viral infections. Thus, the development of IgG against SARS-CoV-2 in the course of COVID-19 might not be a simple sign of viral clearance and developing protection against the virus. On the contrary, due to cross-reaction to related coronavirus strains from earlier infections, in certain patients IgG might enhance clinical progression due to ADE. The patient's viral history of coronavirus infection might be crucial to the development of the current infection with SARS-CoV-2. Furthermore, it poses a note of caution when treating COVID-19 patients with convalescent sera.

Likelihood Ratios as Value Proposition for Diagnostic Laboratory Tests.

The clinical and health economic value of clinical laboratory diagnostics has been debated increasingly in recent years without resulting in practical recommendations for measuring the effectiveness of diagnostic tests. One way to achieve such a goal could be to enrich the mere data of laboratory test results with additional information about their likelihood ratios for diagnosis. The diagnostic significance of test results can be judged subjectively based on the experience of the treating physician or expressed objectively in the form of likelihood ratios. The provision of likelihood ratios by the laboratory would increase the impact of laboratory diagnostics in healthcare and thus have positive economic value. Consequently, likelihood ratios should be taken into account in reimbursement strategies.

A simplified method to approximate a ROC curve with a Bézier curve to calculate likelihood ratios of quantitative test results.

In order to calculate likeli hood ratios (LR) values for quantitative test results, a distribution-independent algorithm based on Bézier curves is proposed. Receiver operating characteristic (ROC) analysis provides the LR as the slope of the tangent to the ROC curve at the point corresponding to the test result. • Here, we make use of cubic Bézier curves defined by Bernstein polynomials of degree 3. • A simplified method to adjust a Bézier curve to a ROC curve is presented • The crucial advantage of this procedure is that Bézier curves are constructed by tangents to the ROC curve, whose slopes immediately provide the LR of a specific point on the curve.

Optimization of serologic diagnosis of celiac disease in the pediatric setting.

BACKGROUND: The clinical presentation of celiac disease (CD) varies between children. The objective of this study was to document the pre-test probability for CD based on symptoms and routine laboratory test and to evaluate the performance of two IgA anti-tissue transglutaminase (tTG) assays. We critically reviewed the concept of using multiples of the manufacturer's upper limit of normal (ULN), as proposed in the ESPGHAN guidelines (if IgA tTG is >10 times ULN, no biopsy is needed). METHODS: The retrospective study included 91 children with newly diagnosed CD and 605 controls (<16 years). All underwent upper endoscopy with small bowel biopsies. Four laboratory parameters and 16 symptoms were registered. All patients were tested for IgA anti-tTG antibodies with assays from Inova Diagnostics and Thermo Fisher Scientific. RESULTS: Some combinations of clinical symptoms and laboratory parameters had a high pre-test probability for CD, such as (combinations of) anorexia, failure to thrive, low ferritin level and elevated AST. The diagnostic performance of both IgA anti-tTG assays was excellent and comparable (no difference in ROC curve area under the curve). At a threshold that corresponds to a specificity of 100% (5 times ULN for Inova Diagnostics and 2 times ULN for Thermo Fisher), the sensitivity was 82% for both assays. At the 10 times ULN threshold, the sensitivity differed between the assays (77% vs. 57%), indicating that such threshold does not completely align interpretation across companies. CONCLUSIONS: Our study showed that some combinations of symptoms and aberrant laboratory parameters had a high pre-test probability. The use of the ESPGHAN non-biopsy approach could reduce small bowel biopsies, but thresholds for IgA-tTG levels are not aligned across assays and should be based on predefined likelihood ratios or specificity.

Application of Bézier Curves for Calculating Likelihood Ratios for Plasma Amyloid-ß Biomarkers for Alzheimer's Disease.

Introduction: Alzheimer's disease, being the most frequent cause of dementia in elderly people, still is difficult to diagnose and to predict its occurrence. The clinical application of biomarkers for diagnosis of Alzheimer's disease has been restricted so far to the analysis of proteins in the cerebrospinal fluid like amyloid ß1-42 and p-tau. However, in a recently published nature letter it has been shown that the high-performance measurement of amyloid-ß in plasma alone could provide a method well suited for a broad clinical application. The study uses ROC analysis to evaluate the clinical significance of the method but it does not provide likelihood ratios (LR) of the measured results. Methods: In this article, a newly developed method is used to calculate LRs for any measurement result of a study by approximation of the ROC curves using Bézier curves. Such LRs provide an estimation of the clinical significance of any particular test result by applying Bayes' theorem: Pretest odds for disease multiplied by the LR of the test result give the posttest odds. Results: The application of the Bézier curve approximation to the data of the plasma amyloid-ß study is demonstrated. To generalize the calculation of LRs for all test results, a relation between the test results and the points on the Bézier curve with their LRs is established. Discussion: The application of Bézier curves in ROC analysis allows calculating LRs for all individual test results when measuring amyloid-ß biomarkers for Alzheimer's disease.

Likelihood ratios of quantitative laboratory results in medical diagnosis: The application of Bézier curves in ROC analysis.

Receiver operating characteristic (ROC) analysis is widely used to describe the discriminatory power of a diagnostic test to differentiate between populations having or not having a specific disease, using a dichotomous threshold. In this way, positive and negative likelihood ratios (LR+ and LR-) can be calculated to be used in Bayes' way of estimating disease probabilities. Similarly, LRs can be calculated for certain ranges of test results. However, since many diagnostic tests are of quantitative nature, it would be desirable to estimate LRs for each quantitative result. These LRs are equal to the slope of the tangent to the ROC curve at the corresponding point. Since the exact distribution of test results in diseased and non-diseased people is often not known, the calculation of such LRs for quantitative test results is not straightforward. Here, a simple distribution-independent method is described to reach this goal using Bézier curves that are defined by tangents to a curve. The use of such a method would help in standardizing quantitative test results, which are not always comparable between different test providers, by reporting them as LRs for a specific diagnosis, in addition to, or instead of, quantities such as mg/L or nmol/L, or even indices or units.

Age-Related Macular Degeneration: A Connection between Human Herpes Virus-6A-Induced CD46 Downregulation and Complement Activation?

Viruses are able to interfere with the immune system by docking to receptors on host cells that are important for proper functioning of the immune system. A well-known example is the human immunodeficiency virus that uses CD4 cell surface molecules to enter host lymphocytes and thereby deleteriously destroying the helper cell population of the immune system. A more complicated mechanism is seen in multiple sclerosis (MS) where human herpes virus-6A (HHV-6A) infects astrocytes by docking to the CD46 surface receptor. Such HHV-6A infection in the brain of MS patients has recently been postulated to enable Epstein-Barr virus (EBV) to transform latently infected B-lymphocytes in brain lesions leading to the well-known phenomenon of oligoclonal immunoglobulin production that is widely used in the diagnosis of MS. The cellular immune response to HHV-6A and EBV is one part of the pathogenic mechanisms in MS. A more subtle pathogenic mechanism can be seen in the downregulation of CD46 on astrocytes by the infecting HHV-6A. Since CD46 is central in regulating the complement system, a lack of CD46 can lead to hyperactivation of the complement system. In fact, activation of the complement system in brain lesions is a well-known pathogenic mechanism in MS. In this review, it is postulated that a similar mechanism is central in the development of age-related macular degeneration (AMD). One of the earliest changes in the retina of AMD patients is the loss of CD46 expression in the retinal pigment epithelial (RPE) cells in the course of geographic atrophy. Furthermore, CD46 deficient mice spontaneously develop dry-type AMD-like changes in their retina. It is also well known that certain genetic polymorphisms in the complement-inhibiting pathways correlate with higher risks of AMD development. The tenet is that HHV-6A infection of the retina leads to downregulation of CD46 and consequently to hyperactivation of the complement system in the eyes of susceptible individuals.

Multiple sclerosis: an example of pathogenic viral interaction?

A hypothesis is formulated on viral interaction between HHV-6A and EBV as a pathogenic mechanism in Multiple Sclerosis (MS). Evidence of molecular and genetic mechanisms suggests a link between HHV-6A infection and EBV activation in the brain of MS patients leading to intrathecal B-cell transformation. Consequent T-cell immune response against the EBV-infected cells is postulated as a pathogenic basis for inflammatory lesion formation in the brain of susceptible individuals. A further link between HHV-6A and EBV involves their induction of expression of the human endogenous retrovirus HERV-K18-encoded superantigen. Such virally induced T-cell responses might secondarily also lead to local autoimmune phenomena. Finally, research recommendations are formulated for substantiating the hypothesis on several levels: epidemiologically, genetically, and viral expression in the brain.

Conceptual Spaces of the Immune System.

The immune system can be looked at as a cognitive system. This is often done in analogy to the neuro-psychological system. Here, it is demonstrated that the cognitive functions of the immune system can be properly described within a new theory of cognitive science. Gärdenfors' geometrical framework of conceptual spaces is applied to immune cognition. Basic notions, like quality dimensions, natural properties and concepts, similarities, prototypes, saliences, etc., are related to cognitive phenomena of the immune system. Constraints derived from treating the immune system within a cognitive theory, like Gärdenfors' conceptual spaces, might well prove to be instrumental for the design of vaccines, immunological diagnostic tests, and immunotherapy.

[The concept of reference change values (RCV). Will it supersede reference intervals?]. / Der Referenzbereich ist tot - es lebe der Reference Change Value.

Reference values are generally used to allow a decision on whether a laboratory value is in the normal range or if it mirrors a pathological process. This decision is especially difficult to take, when the pathological process is just starting and the values are relatively close to the normal range. Particularly in this phase, the decision is extremely important. Harris and later on Fraser have realized that there are two variables that contribute to the credibility and significance of a measured analyte. 1. The imprecision of the measurement itself. These values have become relatively low in recent years: they amount to values between 1 and 5 %. 2. The within person biological variability, which can be 100 % or more. Both variables combined yield the "reference change value" (RCV) to define the minimal significant difference between two measurements at different time points. When using this concept, differences between two measurements can be detected before the normal range is exceeded. For any given patient the reference values of a population is actually not of primary concern. It is important to know that his personal data exceed his personal normal range, which is dependent on RCV. For many analytes in clinical chemistry and hematology the use of RCV rather than the normal range as reference improves the decision making process in a clinical setting.

Benefits and risks of IgA in immunoglobulin preparations.

The case of Immunoglobulin A (IgA) in transfusion medicine is unsettled: on one hand IgA is an important component of adaptive immunity and its deficiency may cause disease, on the other its presence in blood products might induce, in rare instances, allergy-like symptoms if not anaphylaxis. The practice with i.v. immunoglobulins currently changes as up to 10% concentrated preparations are given at fast rates hence even trace amounts of IgA contained in these IgG preparations can cause unexpected (side-) effects. Fortunately, the spectrum of sensitive IgA assays, along with anti-IgA screening assays now permits laboratories to narrow down IgA-dependent transfusion reactions to the real cases, in which IgA was the decisive trigger of anaphylaxis, proven or not by the presence of anti-IgA of the IgG or even IgE class. Tolerance to allogenic IgA has recently been reported. The known association of HLA with IgA deficiency (IgAD) has now been completed with an association to the nonsynonymous variant in IFHI1, allowing physicians to more precisely spot recipients at risk for an IgA-dependent transfusion reaction. Our review, along with our own experience here in Switzerland, allows us to conclude that IgA is a beneficial antibody rather than an allergen to be placed at the end of the list of non-infectious transfusion complications such as TRALI, febrile non-hemolytic reactions, purpura or volume overload.

The role of compartment penetration in PI-monotherapy: the Atazanavir-Ritonavir Monomaintenance (ATARITMO) Trial.

OBJECTIVES: To limit exposure to anti-HIV drugs and minimize risk of long-term side effects, studies have looked at the possibility of simplified maintenance strategies. Ritonavir-boosted protease-inhibitor (PI)-monotherapies are an attractive alternative, but limited compartmental penetration of PI remains a concern. DESIGN: Non-comparative 24-week pilot study. METHOD: Ritonavir-boosted atazanavir (ATV/r) monotherapy administered to fully suppressed patients (>3 month HIV RNA < 50 copies/ml). Plasma was obtained every 4 weeks and cerebrospinal fluid (CSF) and semen at W24. RESULTS: Two patients (7%) failed ATV/r monotherapy. One patient was subsequently identified as a protocol violator since he had a previous history of treatment failure under indinavir. The second patient deliberately decided to stop treatment after W20. Excluding failing patients, individual measurements of HIV RNA in patients having occasional viral 'blips' was found in five patients. At W24, 3/20 patients had elevated viral loads in CSF (HIV RNA > 100 copies/ml), and 2/15 in semen, despite viral suppression in plasma (< 50 copies/ml). Samples with elevated HIV RNA (> 500 copies/ml) in CSF were all wild type. The mean ATV drug concentration ratio (CSF/blood, n = 22) was 0.9%. Indicators of altered immune activation (CD8CD38 C-reactive protein) remained unchanged. CONCLUSION: This study supports previous results indicating the potential use of PI-based mono-maintenance therapies. However, our results in CSF cautions against the uncontrolled use of PI-based monotherapies.

Cross-sectional survey on hantavirus seroprevalence in Canton St. Gallen, Switzerland.

BACKGROUND AND OBJECTIVES: In 2002 the first endemic hantavirus infection in Switzerland was detected only by chance following a broad spectrum of diagnostics. This raised the question, whether Hantavirus infection should be included in the differential diagnosis of febrile illness of patients in Switzerland. In order to estimate the frequency of hantavirus infections in Switzerland, this survey on hantaviral seroprevalence was conducted in the Canton St. Gallen. METHODS: A total of 1693 sera from farmers, forestry workers, and young soldiers as well as blood donors, as a cross-section of the average adult population of the Canton St. Gallen, were screened for hantavirus-specific antibodies by a microsphere-based assay. All volunteers with positive screening results obtained a questionnaire for assessment of details of previous rodent encounter and illnesses compatible with hantavirus infection. RESULTS: This first survey on hantavirus-specific IgG in populations of eastern Switzerland revealed low seroprevalence-rates not significantly different among populations with higher risk for hantavirus infection (0.0%-1.9%) and the average adult population (0.5%). CONCLUSIONS: As hantavirus infections among different populations are rare, and no evidence for hantaviral nephropathy could be found, serological investigation of suspected endemic hantavirus infection in eastern Switzerland should be confined to patients with acute nephropathy and/or a history of recent rodent encounter.

Diagnostic significance of intrathecally produced herpes simplex and varizella-zoster virus-specific antibodies in central nervous system infections.

BACKGROUND AND OBJECTIVES: The optimal strategy for the diagnosis of herpes simplex virus (HSV) and varizella-zoster virus (VZV) disease of the central nervous system is the detection of viral DNA by polymerase chain reaction assay (PCR) in cerebrospinal fluid (CSF) and the examination of intrathecal production of specific antibodies. However, in acute neurological disease caused by either HSV or VZV, dual intrathecal synthesis of HSV-1, 2- as well as VZV-specific antibodies may be detectable and thus can hamper accurate aetiological diagnosis. This paper illustrates such equivocal findings in two case reports, investigates their frequency and discusses the possible reasons. METHODS: Consecutive CSF/serum pairs of two patients with central nervous system (CNS) disease were tested by HSV-1-, HSV-2-, and VZV-specific PCR and by different serological assays for detection of neurotropic viruses and bacteria. Additionally, the results of microbiological investigations of 1'155 CSF/serum samples were retrospectively analyzed for coincident intrathecal antibody synthesis against HSV-1, 2 and VZV. RESULTS: Although only HSV-1 and VZV-specific DNA was detectable in the CSF of two patients with encephalitis and chronic meningitis, respectively, increasing intrathecal antibody production against both virus species could be demonstrated. Retrospective analysis of 1155 CSF/serum pairs revealed 55 (4.8%) pairs with evidence for intrathecally produced antibodies against either HSV-1, 2 (30/55) or VZV (14/55). Eleven of these 55 (20%) pairs showed intrathecal antibody-production against both virus species. CONCLUSIONS: Patients with CNS infection with HSV and VZV can be diagnosed by detecting intrathecally produced virus-specific antibodies, in addition to virus-specific PCR. However, in an appreciable proportion of patients a correct diagnosis is hampered by coincidentally detected antibodies in CSF against both virus species. Possible reasons for these equivocal findings are given.