Laboratory and clinical practices in antinuclear antibody detection and related antigens: recommendations from a Spanish multicentre survey.

Antinuclear antibodies (ANA) are the most widely used immunological test for the diagnosis of autoimmune diseases. Despite the recommendations of experts, there is some variability in performing and interpreting this test in routine practice. In this context, the Spanish Group on Autoimmune Diseases (GEAI) of the Spanish Society of Immunology (SEI) conducted a national survey of 50 autoimmunity laboratories. Here we report the survey results on ANA testing, detection of related antigens, and our recommendations. The survey showed that most of the participating laboratories use a similar approach for most key practices: 84% perform ANA by indirect immunofluorescence (IIF) on HEp-2 cells as the screening methodology while the other laboratories use IIF to confirm positive screens; 90% report ANA test results as either negative or positive with titer and pattern; 86% indicated that the ANA pattern conditioned follow-up testing for specific antigen-related antibodies; and 70% confirm positive anti-dsDNA. However, testing practices were highly heterogeneous for certain items, such as sera dilutions and the minimum time period for repeating ANA and related antigen determinations. Overall, this survey shows that most autoimmune laboratories in Spain use a similar approach but that further standardization of testing and reporting protocols is needed.

Early riser specific immune cell response by delayed-type hypersensitivity in a kidney transplant patient vaccinated against COVID-19.

We present a female kidney transplant patient under conventional immunosuppression therapy. Her humoral immunity study (anti-spike-specific antibodies) was negative after the initial regimen and the third dose of vaccination against COVID-19. The specific ex vivo cellular immune study against spike of SARS-CoV-2 by interferon gamma release assay (IGRA) also remained at non-response levels at different time points despite an optimal non-specific cell immune response assessment. However, the cellular immunity test by delayed-type hypersensitivity (DTH) with spike of SARS-CoV-2 was always positive since the vaccination scheme began. Only after COVID-19 infection has there been a seroconversion of the patient's antibody tests along with IGRA positivity. The use of DTH test to measure the immune response could be a better and earlier parameter of the actual immune status that helps us to predict the immune response in real life. Hybrid immunity combining vaccine and natural infection could be a stronger stimulator of the specific global immune response.

Salmonella Typhi Vaccination Response as a Tool for the Stratification of Risk in Patients with Predominantly Antibody Deficiencies.

Predominantly antibody deficiencies are the most frequent type of primary immunodeficiency (PID). Diagnosis requires evaluation of the immune function by distinguishing the presence or absence of a response against polysaccharide antigens. Salmonella enterica serovar Typhi-based vaccines have proved to be a suitable tool. We studied a group of patients with suspicion of primary immunodeficiency and classified them by final diagnosis. We analyzed the vaccination response to S. Typhi and other immune biomarkers and clinical data. The aim of this study was to classify patients regarding the intensity of their immune response measured as the difference between specific immunoglobulin G levels before and after vaccination and antibody levels in the post-vaccination sample in order to improve clinical decisions regarding follow up and treatment of immunodeficiency patients. We established four groups of response: Non responders (NR), Low responders (LR), Intermediate responders (IR), and High responders (HR), where we found differences in IgG, IgG1, IgG2, IgG4, IgA, IgA1, IgA2, and IgM, and where the finally achieved diagnosis was also different and corresponding to the level of vaccination response.

Long Term Cell Immune Response to COVID-19 Vaccines Assessment Using a Delayed-Type Hypersensitivity (DTH) Cutaneous Test.

OBJECTIVE: As we progress with mass vaccination against SARS-CoV-2, there are key questions about the immunogenicity of COVID-19 vaccines that still are not answered. Conventional methods to measure cellular immune responses are complex and expensive in a pandemic situation. PATIENTS AND METHODS: Forty healthy healthcare workers accepted to participate during the vaccination schedule with a mRNA vaccine against SARS-CoV-2. Measurement of Delayed-Type Hypersensitivity (DTH) cutaneous response after intradermal test of protein S of SARS-CoV-2 at day 35 and day 200 was performed. At the same time, a specific anti-RBD IgG using a classic ELISA before vaccination, and on days 0, 35, and 200 was performed. RESULTS: All 40 individuals had a positive DTH skin response at day 35, whereas 39 participants had a positive skin test at day 200. Moreover, although all 40 individuals showed a positive humoral response of specific IgG against spike protein at day 35, with most of them having significantly lower levels at day 200. CONCLUSION: DTH could be proposed as an ideal and easy method to predict cellular immunity response to mRNA vaccines 200 days after starting an immunization schedule with mRNA vaccine for COVID-19.

Easy approach to detect cell immunity to COVID vaccines in common variable immunodeficiency patients.

BACKGROUND: Patients with primary antibody deficiencies, such as Common Variable Immunodeficiency (CVID), have some problems to assess immune response after coronavirus disease (COVID) vaccination. Cutaneous delayed-type hypersensitivity (DTH) has the potential to be used as a useful, simple, and cheaper tool to assess T-cell (T lymphocyte) function. METHODS: Seventeen patients with CVID, a rare disease, received two doses of the mRNA-based Pfizer-BioNTech COVID-19 vaccine. Humoral Immune Response (HIR) was determined by measuring specific immunoglobulin G (IgG) antibodies, and Cellular Immune Response (CIR) was evaluated using an ex vivo interferon-gamma release assay (IGRA) and in vivo by DTH skin test. RESULTS: Two weeks after the second dose of the vaccine, 12 out of 17 CVID patients have high optical density (OD) ratios of specific anti-spike protein (S) IgG whereas five patients were negative or low. Ex vivo CIR was considered positive in 14 out of 17 S1-stimulated patients. Unspecific stimulation was positive in all 17 patients showing no T-cell defect. A positive DTH skin test was observed in 16 CVID patients. The only patient with negative DTH also had negative ex vivo CIR. CONCLUSIONS: The use of DTH to evaluate CIR was validated with an optimal correlation with the ex vivo CIR. The CIR after vaccination in patients with antibody deficiencies seems to have high precision and more sensitivity to antibodies-based methods in CVID. CLINICAL IMPLICATIONS: There is a remarkable correlation between cutaneous DTH and ex vivo IGRA after COVID vaccination. A COVID-specific skin DTH test could be implemented in large populations. CAPSULE SUMMARY: Cutaneous delayed-type hypersensitivity has the potential to be used as a useful, simple, and cheaper tool to assess T-cell functioning.

A review of hypersensitivity methods to detect immune responses to SARS-CoV-2.

The investigation of the immune response after SARS-CoV-2 infection has been the goal of many researchers worldwide. The study of humoral immune responses and in vitro T cell production after infection requires the obtaining of individualized blood samples to test the presence of antibodies or activated T cells specific for the virus. In vitro T cell studies are especially troublesome due to the need for more specialized resources often outside the daily routine of clinical laboratories. For this reason the development of a simple and objective method to achieve these T cell studies is needed. In this manuscript we reviewed the hypersensitivity reactions, the theoretical basis and the historical background of delayed type hypersensitivity (DTH) which uses the principles of use of this test in the clinical setting for the past century. In the second part of the review, we focus on COVID adaptive immune responses, to understand the differences and challenges offered by this new application of DTH to investigate immune responses elicited after infection. In the last part of the review a vision provided for the use of this test to investigate the immunogenicity elicited by the vaccines. In our opinion, the clinical guidelines of immune assessment of SARS-CoV-2-infected or vaccinated individuals should include this simple and low-cost test to measure T-cell immunity. Rationale and improved vaccination schemes could be obtained after its implementation in the routine assessment of immunity in this pandemic situation.

Easy approach to detect cell immunity to COVID vaccines in common variable immunodeficiency patients

Background Patients with primary antibody deficiencies, such as Common Variable Immunodeficiency (CVID), have some problems to assess immune response after coronavirus disease (COVID) vaccination. Cutaneous delayed-type hypersensitivity (DTH) has the potential to be used as a useful, simple, and cheaper tool to assess T-cell (T lymphocyte) function.Methods Seventeen patients with CVID, a rare disease, received two doses of the mRNA-based Pfizer-BioNTech COVID-19 vaccine. Humoral Immune Response (HIR) was determined by measuring specific immunoglobulin G (IgG) antibodies, and Cellular Immune Response (CIR) was evaluated using an ex vivo interferon-gamma release assay (IGRA) and in vivo by DTH skin test.Results Two weeks after the second dose of the vaccine, 12 out of 17 CVID patients have high optical density (OD) ratios of specific anti-spike protein (S) IgG whereas five patients were negative or low. Ex vivo CIR was considered positive in 14 out of 17 S1-stimulated patients. Unspecific stimulation was positive in all 17 patients showing no T-cell defect. A positive DTH skin test was observed in 16 CVID patients. The only patient with negative DTH also had negative ex vivo CIR.Conclusions The use of DTH to evaluate CIR was validated with an optimal correlation with the ex vivo CIR. The CIR after vaccination in patients with antibody deficiencies seems to have high precision and more sensitivity to antibodies-based methods in CVID.Clinical Implications There is a remarkable correlation between cutaneous DTH and ex vivo IGRA after COVID vaccination. A COVID-specific skin DTH test could be implemented in large populations.Capsule Summary Cutaneous delayed-type hypersensitivity has the potential to be used as a useful, simple, and cheaper tool to assess T-cell functioning (AU)

Optimizing a Protocol to Assess Immune Responses after SARS-CoV-2 Vaccination in Kidney-Transplanted Patients: In Vivo DTH Cutaneous Test as the Initial Screening Method.

Previously, the delayed-type hypersensitivity (DTH) cutaneous test with the spike protein of SARS-CoV-2 has been shown to be a simple in vivo method to measure T-cell functionality after natural infection and in vaccinated individuals. METHODS: Twenty-five kidney-transplanted recipients were immunized with two doses of the mRNA-based Pfizer-BioNTech COVID19 vaccine three weeks apart. Cell-immune response (CIR) was evaluated ten weeks later using an in vivo DTH skin test and in vitro with an interferon gamma release assay (IGRA). Humoral Immune Response (HIR) was determined by the measurement of specific IgG anti-S1 SARS-CoV-2. RESULTS: Ten weeks after the second dose of the vaccine, 23 out of 25 transplanted patients had a positive DTH skin test, while in vitro CIR was considered positive in 20 patients. Unspecific stimulation was positive in all 25 patients, showing no T-cell defect. Seven out of twenty-five patients had a negative specific anti-spike IgG. CIR was positive in all immune-competent control patients. CONCLUSIONS: DTH is a useful, simple, and cheaper tool that can be used to assess cellular immune response, with an excellent correlation with the in vitro CIR. CIR assessment after vaccination in these immunocompromised patients is an excellent complement to HIR-based methods. This skin test could be used if classical in vitro methods cannot be applied.

The Beauty of Simplicity: Delayed-Type Hypersensitivity Reaction to Measure Cellular Immune Responses in RNA-SARS-Cov-2 Vaccinated Individuals.

BACKGROUND: Monitoring cellular immune responses elicited in vaccinated individuals is highly complicated. METHODS: 28 individuals participated during the vaccination process with 12 BNT162b2 mRNA (Pfizer) vaccine. Specific anti-RBD IgG using a classic ELISA was performed in days 10 and 20 (after one dose of the vaccine) and on day 35 (after two vaccine doses) in serum samples of all participants. In parallel, DTH (delayed-type hypersensitivity) Skin Test using S protein was performed before (11/28) and after two doses (28/28) of the vaccine. RESULTS: 6/28 individuals were considered positive for the specific anti-RBD IgG positive at day 10, whereas all 28 individuals were positive at day 20. Moreover, 28/28 individuals increased the OD ratios at day 36 (2 doses). DTH cutaneous test was performed on 11/28 participants at day 20 (1 dose) showing 8/11 a positive reaction at 12 h. DTH of all participants was performed on day 36 (2 doses), showing 28/28 positive reactions at 12 h. CONCLUSION: This report describes the first publication of the results obtained using an in vivo method, the classical DTH response to the Spike protein to assess T-cell immune responses in vaccinated individuals. This affordable and simple test would help to answer basic immunogenicity questions on large-scale population vaccine studies.

A novel application of delayed-type hipersensitivity reaction to measure cellular immune response in SARS-CoV-2 exposed individuals.

OBJECTIVE: To understand the anti-virus adaptive immune response occurring during SARS-Cov-2 infection is necessary to have methods to investigate cellular and humoral components. The goal of this study has been to investigate the utility of a specific spike-DTH test using a coronavirus recombinant protein in COVID-19 patients. METHODS: DTH studies were performed by intradermal injection of a commercial recombinant spike protein from SARS-CoV-2 along with conventional serology studies. RESULTS: Fifty-one COVID-19 patients were studied showing 84,3% of concordance with spike-DTH and anti-RBD-IgG. Spike-DTH was superior to identify seven more COVID-19 individuals. A high specificity was found with no positive spike DTH reactions in the non-sick individuals. The skin test also showed more stable results over time while specific anti-RBD-IgG decreased gradually. Clinical severity groups also showed a progressive gradient of larger positive spike-DTH. CONCLUSION: Specific spike DTH test seems to be an easy method to study cell immune response.

Isolated Ro52 Antibodies as Immunological Marker of a Mild Phenotype of Undifferentiated Connective Tissue Diseases.

The term undifferentiated connective tissue disease (UCTD) is used to describe undiagnosed patients that do not fulfill classification criteria for definite connective tissue disease (Systemic Lupus, Systemic Sclerosis, Sjögren Syndrome, and Dermatomyositis/Polymyositis). It is important to find serological markers as predictors of the evolution or severity of these diseases. The objective of this retrospective study was to investigate if there was a milder subgroup of UCTD with a special clinical profile consisting only in the presence of anti-Ro52 autoantibodies. Immunological and clinical records of 62 patients attending the hospital during 30 months were studied. Results showed a target population formed by mostly women, aged between 40 and 80 years at the moment of the study, with a registered age of onset between 40 and 60 years. Speckled pattern was the most frequent pattern found by indirect immunofluorescence. Given the obtained results and keeping in mind possible limitations because of sample size, isolated positive anti-Ro52 autoantibodies seem to lead to a benign effect in terms of evolution of the disease. As a future objective, the follow-up of these patients should be necessary to investigate new clinical symptoms, serological markers, or development of a definite connective tissue disease over time.

Role of specific IgE to ß-lactoglobulin in the gastrointestinal phenotype of cow's milk allergy.

RATIONALE: The prevalence of many phenotypes of food allergy is increasing. Specific gastrointestinal (GI) phenotype of food allergy (GI allergy) is also increasing but it is difficult to know the prevalence because of many entities. METHODS AND RESULTS: A 1 year retrospective study of pediatric patients complaining exclusively gastrointestinal symptoms after cow's milk consumption and at least one positive specific IgE (sIgE) to cow's milk (CM) proteins (CMP) was done (n = 39). The most prevalent symptom was abdominal cramps in 35 patients (90 %), discomfort or abdominal distention in 30 patients (75 %), diarrhea in 10 patients (25 %) and constipation in 5 patients (12 %). IgA anti-transglutaminase antibodies were absent and lactose intolerance was ruled out in all patients. Average of total IgE on this group was 288 UI/ml. sIgE against ß-lactoglobulin was the dominant with an average of 4.14 kU/l. sIgE to casein (CAS), which is the dominant protein in systemic anaphylaxis was 1.74 kU/l; sIgE to α-lactoalbumin, the other whey protein, was 0.83 kU/l and sIgE levels to CM were 0.78 kU/l. The quotient sIgE CAS/sIgE ß-lactoglobulin in these patients was always lower than 1. Patients experienced an improvement of their symptoms after a CM free diet. An open oral challenge with CM did mimic their initial symptoms in all patients. However, the open oral challenge with dairy products was well tolerated. CONCLUSIONS: Patients with a specific phenotype of GI allergy with CM have specific IgE against ß-lactoglobulin, as a dominant sIgE. These patients could beneficiate of a diet with dairy products.

Downregulation of angiogenesis factors, VEGF and PDGF, after rapid IgE desensitization and oral immunotherapy in children with food allergy.

BACKGROUND: Angiogenesis has a key role in several conditions and is regulated by several factors such as the platelet-derived growth factor (PDGF) or the vascular endothelial growth factor (VEGF). The goal of this study was to investigate the possible role of PDGF and VEGF in a group of patients with severe food allergy. METHODS: We design a prospective longitudinal study (n = 30) with patients with persistent cow's milk proteins (CMP) allergy. After achieving a CMP rush desensitization protocol, a clinical followup including SPT and blood samples to determine sIgE, protein levels, PDGF, and VEGF-A and a panel of the most representative Th1, Th2, Treg, and Th17 cytokines were also monitored. RESULTS: Baseline levels of PDGF and VEGF in the CMP allergic patients (1170 pg/mL and 253 pg/mL) were different compared to those nonallergic CMP control subjects (501 pg/mL and 108 pg/mL). Both PDGF and VEGF were significantly downregulated (P < 0.05) 6 months after completion of the CMP desensitization process and remained significantly decreased 12 months later. CONCLUSION: The present study shows a significant increase of PDGF and VEGF in anaphylaxis suffering children compared to a control group. Interestingly, both VEGF and PDGF were significantly downregulated after completing a full CMP rush IgE desensitization.

T cell activity in successful treatment of chronic urticaria with omalizumab.

Omalizumab, a humanized monoclonal anti-IgE antibody has the potential to alter allergen processing. Recently, it has been postulated the assessment of PHA-stimulated adenosine triphosphate (ATP) activity as maker of CD4+ T cells activity in peripheral blood cells. We present the case report of a 35-year-old woman with a history of chronic idiopathic urticaria and angioedema of 8 years of development with poor response to treatment. The patient was partially controlled with cyclosporine at doses of 100 mg/12 h. However, she was still developing hives daily. Finally treatment with omalizumab was started at dose of 300 mg every 2 weeks. The patient experienced a decrease in urticarial lesions 2 days after starting therapy. We also evaluated the effects of omalizumab therapy on the activity of peripheral blood CD4+ T cells from the patient, in order to determine the potential modification of anti-IgE therapy on the process of antigen presentation-recognition. Activity of CD4+ cells by ATP release was clearly increased demonstrating an enlarged CD4 activity. Omalizumab may be useful in the treatment of severe chronic urticaria. ATP activity of peripheral blood CD4+ T cells might be a non-subjective method to assess Omalizumab activity.

Impact on allergic immune response after treatment with vitamin A.

BACKGROUND: Vitamin A may have some influence on the immune system, but the role in allergy modulation is still unclear. OBJECTIVE: To clarify whether high levels of retinoic acid (RA) affects allergic response in vivo, we used a murine experimental model of airway allergic disease. METHODS: Ovalbumin (OVA)-immunization/OVA-challenge (OVA/OVA) and house dust mite (HDM)-immunization/HDM-challenge (HDM/HDM) experimental murine models of allergic airway disease, using C57Bl.10/Q groups of mice (n = 10) treated subcutaneously with different concentrations of all-trans RA (0, 50, 500 and 2,500 ug) every 2-days were used to assess the allergic immune response. RESULTS: Levels of total and specific-IgE in sera were increased in all groups of RA treated OVA/OVA and HDM/HDM mice. Percentage and total amount of recruited eosinophil in airways by bronchoalveolar lavage fluid (BALF) were significantly enhanced in groups treated with 50, 500 and 2,500 ug of RA compared to non-treated mice. However, the group of mice treated with 2,500 ug had less eosinophil recruitment than the other two groups (50 and 500 ug). In parallel, levels of IL-5 and total IgE in BALF were also significantly diminished in the group treated with 2,500 ug compared to the other 2 groups (50 and 500 ug). Finally, total lung resistance was decreased in group treated with 2,500 ug compared to non-treated mice. CONCLUSION: Our results suggest that retinoic acid directly enhances allergic response in vivo, but in higher doses may produce of immune suppression.

Large Evaluation of Anti-Cardiolipin and anti-Beta2Glycoprotein I Assays: Results from the AutoimmunityWorkshop of the Spanish Society of Immunology

Despite their clinical utility and the importance that laboratory testshave in APS diagnosis, probably the most important drawback of suchtests is the elevated intra- and inter-laboratory variation. The aim of thepresent work was to assess the multilaboratory performance of aCL (..) (AU)

A pesar de la indudable utilidad clínica y de la importancia de laspruebas de laboratorio en el diagnóstico del síndrome antifosfolípido(APS), probablemente el mayor defecto de dichas pruebas es su elevadavariabilidad intra- e inter-laboratorio. El objetivo del presente trabajo fueevaluar el comportamiento de los ensayos (..) (AU)

Anti-idiotypic Immunotherapy in follicular lymphoma patients: results of a long follow-up study.

A specific immune response against the individual idiotypic determinants of clonal immunoglobulins can be elicited in patients with B-cell malignancies. We analyze the clinical outcome and the presence of tumor cells in the blood of 8 patients with follicular lymphoma, vaccinated with autologous idiotype protein from their lymphoma cells. After a median follow-up of 90 months (range: 54 to 128), all patients, except 1, remain in complete clinical remission and 5 are in complete molecular remission. Our results suggest that idiotype vaccination induces long-lasting clinical and molecular remissions and constitutes a potential curative treatment in follicular lymphoma patients.