Falsely low immunoglobulin (Ig)G4 in routine analysis: how not to miss IgG4 disease.

Immunoglobulin (Ig)G4 disease can have apparently 'normal' levels of IgG4 due to antigen excess conditions. IgG4 measurement therefore appears falsely low. UK National External Quality Assurance Scheme (UK NEQAS) data and other reports have suggested that this problem occurred despite pre-existing antigen excess detection steps. To determine the clinical relevance of the problem, we examined the prevalence and characteristics of prozoning in our laboratory and patient cohorts. We establish that the prevalence of raised IgG4 in routine IgG4 analysis is low (< 1%) using one of the two routine methods in use in the United Kingdom. We show that subsequent assay modification appears to have reduced the likelihood of misleading readings. However, the original version of the assay prozoned to low levels (below 0·64 g/l) in 41% of high IgG4 samples in our patients. This may explain the previous reports of low sensitivity of raised IgG4 for IgG4RD, and predictive values should be re-evaluated in this disease using modified prozone-resistant protocols. All laboratories providing IgG4 measurements should verify that their assays are fit for the clinical quality requirement of detection raised IgG4 levels and must verify the upper limit of their reference ranges and freedom from prozoning.

Immunologic assessment and KMT2D mutation detection in Kabuki syndrome.

Kabuki or Niikawa-Kuroki syndrome (KS) is a rare disorder with multiple malformations and recurrent infections, especially otitis media. This study aimed to investigate the genetic defects in Kabuki syndrome and determine if immune status is related to recurrent otitis media. Fourteen patients from 12 unrelated families were enrolled in the 9-year study period (2005-2013). All had Kabuki faces, cleft palate, developmental delay, mental retardation, and the short fifth finger. Recurrent otitis media (12/14) and hearing impairment (8/14) were also more common features. Immunologic analysis revealed lower memory CD19+ cells (11/13), lower memory CD4+ cells (8/13), undetectable anti-HBs antibodies (7/13), and antibody deficiency (7/13), including lower IgA (4), IgG (2), and IgG2 (1). Naïve emigrant lymphocytes, lymphocyte proliferation function, complement activity, and superoxide production in polymorphonuclear cells were all normal. All the patients had KMT2D mutations and 10 novel mutations of R1252X, R1757X,Y1998C, P2550R fs2604X, Q4013X, G5379X, E5425K, R5432X, R5432W, and R5500W. Resembling the phenotype of common variable immunodeficiency, KS patients with antibody deficiency, decreased memory cells, and poor vaccine response increased susceptibility to recurrent otitis media. Large-scale prospective studies are warranted to determine if regular immunoglobulin supplementation decreases the frequency of otitis media and severity of hearing impairment.

Increased serum IgM, immunodeficiency, and autoimmunity: A clinical series.

BACKGROUND: Primary immunodeficiencies (PIDs) are generally characterized by recurrent infections; however they may be complicated by other clinical disorders such as allergy, autoimmunity, and lymphoproliferation. In particular, autoimmunity may be the first manifestation of the disease in patients with low serum immunoglobulins (Ig) levels. Here we describe a group of patients that share features of immunodeficiency and autoimmunity. MATERIALS AND METHODS: All patients went through a complete T and B cell subset characterization and a B cell function analysis in the peripheral blood by flow-cytometry. B cell proliferation and plasma cell differentiation was measured, in vitro, after CpG stimulation for 7 days as previously described. Semi-quantitative PCR analysis for AID and UNG expression as well as serum levels of BAFF were carried out in order to better define the diagnosis. RESULTS: Immunological and molecular analysis did not lead to the identification of known molecular defect typical of Hyper IgM syndrome. A comparative study of the peripheral blood B cell subsets between patients and healthy donors showed that in patients with autoimmune manifestations all circulating B cells expressed high amounts of surface IgM. CONCLUSIONS: These results suggest that the increased IgM expression on circulating B cells, reflecting B cell activation, might identify a clinical condition characterized by hyper IgM serum levels of unknown molecular defects, associated with susceptibility to infections and autoimmunity.

Dysgammaglobulinemia Associated With Glu349del, a Hypomorphic XIAP Mutation.

BACKGROUND: X-linked lymphoproliferative syndrome type 2 is a rare hereditary immunodeficiency caused by mutations in the XIAP gene. This immunodeficiency frequently results in hemophagocytic lymphohistiocytosis, although hypogammaglobulinemia and dysgammaglobulinemia are also common. OBJECTIVE: We identified 17 patients from 12 Japanese families with mutations in XIAP. The Glu349del mutation was observed in 3 patients, each from a different family. Interestingly, these patients exhibited dysgammaglobulinemia but not hemophagocytic lymphohistiocytosis. We conducted an immunological study of patients carrying Glu349del and other mutations to elucidate the pathogenic mechanisms of dysgammaglobulinemia in patients with mutations in the XIAP gene. PATIENTS AND METHODS: We performed an immunological study of 2 patients carrying the Glu349del mutation and 8 patients with other mutations. RESULTS: Flow cytometry showed that the percentage of memory B cells in patients with a mutation in XIAP was lower than that observed in the healthy controls. The patients with the Glu349del mutation had a lower percentage of memory B cells than those with other mutations. Ig production was reduced in patients with the Glu349del mutation. Increased susceptibility to apoptosis was observed in the patients with other mutations. Susceptibility to apoptosis was normal in patients with Glu349del. Microarray analysis indicated that expression of Ig-related genes was reduced in patients with the Glu349del mutation and that the pattern was different from that observed in the healthy controls or patients with other mutations in XIAP. CONCLUSIONS: Patients carrying the Glu349del mutation in the XIAP gene may have a clinically and immunologically distinct phenotype from patients with other XIAP mutations. The Glu349del mutation may be associated with dysgammaglobulinemia.

Recurrent respiratory infections, specific antibody deficiencies, and memory B cells.

OBJECTIVES: To investigate the immunological phenotypes detected in children with recurrent upper and lower respiratory infections that have normal total immunoglobulin concentrations. METHODS: A cohort of over 60 children with recurrent respiration infections was evaluated for specific antibody deficiencies (SAD) and for memory B-cell abnormalities. A control group of children without recurrent infections was also evaluated. Evaluation included a detailed history of immunizations with pneumococcal vaccines; determination of IgM, IgG, IgA, and IgE concentrations; measurement of anti-pneumococcal polysaccharide antibody levels by ELISA and expression of CD27, IgD, and IgM on peripheral CD19(+)B cells by flow cytometry to determine the proportions of naive, IgM-memory B cells, and class-switched memory B cells. RESULTS: Patients were classified as having a SAD to either pure polysaccharides (PPV-SAD) or to conjugate polysaccharides (PCV-SAD) based on the number of polysaccharides to which they developed an adequate antibody response. A normal response to only 2 or fewer of 7 PCV or PPV serotypes was considered as evidence of SAD. Forty-one patients without SAD and 26 with SAD were identified. IgM-memory B cells were low in 3 of 41 patients without SAD; in 3 of 5 PPV-SAD patients; and in 10 of 21 PCV-SAD patients. Class-switched memory B cells were low in 19 of 41 patients without SAD; in all 5 patients with PPV-SAD; and in 11 of 21PCV-SAD patients. CONCLUSIONS: Patients with recurrent infection with or without SAD may have low IgM- and/or class-switched memory B cells. Ongoing research aims to determine the prognostic implications of these differences in patients with SAD.

Significant differences in B-cell subpopulations characterize patients with chronic graft-versus-host disease-associated dysgammaglobulinemia.

Manifestations of chronic graft-versus-host disease (cGVHD) can resemble those seen in immunodeficiency states and autoimmune disorders. Reports by us and others suggest an involvement of B cells in the pathogenesis of cGVHD. We investigated B-lymphocyte subpopulations in cGVHD cohorts defined by serum immunoglobulin G (IgG) levels to characterize novel biomarkers for impairment of humoral immunity after allogeneic hematopoietic stem cell transplantation. Seventy-six patients were enrolled a median of 46 months after hematopoietic stem cell transplantation. The hypogammaglobulinemia group had significantly diminished CD19(+) B cells (165 vs 454 vs 417 × 106L) with elevated CD19(+)CD21(low) immature (16.5%, 7.7%, and 9.1%) and CD19(+)CD21(int-high)CD38(high)IgM(high) transitional (10.5% vs 4.2% vs 6.3%) B-cell proportions compared with the normogammaglobulinemia and hypergammaglobulinemia groups. CD19(+)CD10(-)CD27(-)CD21(high) naive B cells were highly elevated in all patients with cGVHD. CD19(+)CD27(+)IgD(+) non-class-switched (4 vs 12 vs 11 × 106/L) and class-switched (7 vs 35 vs 42 × 106/L) memory B cells were significantly lower in the hypogammaglobulinemia group compared with the others. Besides significantly higher B-cell activation factor/B-cell ratios, significantly more cGVHD patients with hypergammaglobulinemia had autoantibodies compared with the hypogammaglobulinemia subgroup (68% vs 24%, P = .024). In conclusion, B-cell subpopulations can serve as novel cellular biomarkers for immunodeficiency and autoimmunity indicating different pathogenetic mechanisms of cGVHD and encouraging future prospective longitudinal studies.

Poor specific antibody response immunodeficiency (dysgammaglobulinemia) predates systemic lupus erythematosus.

Poor specific antibody response is a well-known primary immunodeficiency that is related to hypogammaglobulinemia or common variable immunodeficiency (CVID). The co-existence of CVID or hypogammaglobulinemia and systemic lupus erythematosus (SLE) has been rarely described. In all reported cases, the diagnosis of SLE antedates CVID. We report a 15-year-old Saudi girl who was diagnosed with poor specific antibody response at age 6 years in the form of poor or no antibody response and dysgammaglobulinemia. She developed SLE with musculoskeletal and hematological manifestations, positive antinuclear antibody and high anti-dsDNA nine years later. She was treated with rituximab with good response.

Immunoparesis and monoclonal gammopathy of undetermined significance are disassociated in advanced age.

Immunoparesis and a skewed serum free light chain (FLC) ratio are indicators of immune dysfunction predictive of progression from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM). Previous studies have reported increased prevalence of MGUS by age, but no study has examined the relationship between immunoparesis and abnormal FLC ratios in the elderly. We screened 453 older adults (median age, 80 years; range, 65-96) to characterize the patterns of immunoparesis and abnormal FLC ratio in relation to MGUS. We defined MGUS in 4.4% of the subjects; the prevalence was 12.5% among individuals of >90 years. In MGUS (vs. non-MGUS) cases, immunoparesis and abnormal FLC ratios were detected in 70.0% (vs. 49.0%; P = 0.07) and 50.0% (vs. 12.9%; P = 0.0001), respectively. Based on small numbers, MGUS patients with abnormal FLC ratio were borderline (P = 0.07) more likely to have immunoparesis. Overall, the prevalence of immunoparesis varied in a nonlinear fashion, with lowest frequencies in the youngest and oldest groups. Our observed disassociation between MGUS prevalence and impaired immunoglobulin production suggests that separate mechanisms are involved in the development of MGUS and immunoparesis in advanced age. These findings emphasize the need for molecularly defined methods to characterize myeloma precursor states and better predict progression to MM.

[Primary selective IgM deficiency--forgotten primary immunodeficiency?]. / Izolowany niedobór IgM--ignorowany pierwotny niedobór odpornosci?

Immunoglobulin M is a pentamer found in the intravascular compartment and on the surface of B lymphocytes. It is the antibody isotype produced initially in the immune response, and the first immunoglobulin class to be synthesized by a fetus or newborn. IgM antibodies do not cross the placenta. Decreased levels of IgM have been associated with autoimmune disease, several primary immunodeficiency but exist also as selected primary immunodeficiency.

The absence of immunoglobulin D B cell receptor-mediated signals promotes the production of autoantibodies and exacerbates glomerulonephritis in murine lupus.

Immunoglobulin (Ig)D is the major antigen receptor isotype co-expressed with IgM on the surface of most peripheral B cells in mice and humans. However, the biological role of IgD as B cell receptor (BCR) has remained unclear. Previous studies have indicated that IgD may play a role in B cell tolerance. To understand the role of IgD in B cell tolerance and autoimmunity, we have examined the development of autoimmune syndrome in lpr mice deficient for IgD. The present study showed that IgD deficiency did not alter lymphoproliferation and lymphocyte activation in lpr mice. The survival and proliferation of B cells were not affected by the absence of IgD, indicating that IgD BCR-mediated signals do not have an important role in negative selection of autoreactive B cell clones. Interestingly, compared to IgD-competent littermates, lpr mice with IgD deficiency had elevated autoantibody production, increased deposition of immune complex in the kidney and more severe nephritis. Accumulation of abnormal CD4(-) CD8(-) αß(+) T cells was accelerated in IgD(-/-) lpr mice compared to lpr mice. These results suggest that IgD BCR-mediated signals may be involved in the differentiation of autoreactive B cells into plasma cells and abnormal T cell expansion.

Serum Protein Electrophoresis May Be Used as a Screening Tool for Antibody Deficiency in Children and Adolescents.

Background: Patients with antibody deficiency may experience exceptionally long diagnostic delays, increasing the risk of life-threatening infections, end-organ damage, mortality, and health costs. Objective: This study aimed to analyze serum protein electrophoresis and verify the correlation between calculated globulin (CG, total protein minus albumin levels) or electrophoretically determined serum gamma globulin fraction (Gamma) with IgG levels in children and adolescents under 18 years old (yo). Methods: We analyzed serum protein electrophoresis (GC or Gamma) and IgG levels from 1215 children and adolescents under 18 yo, classified into 5 age groups. We verified the correlation between CG or Gamma with serum IgG levels. Results: Serum IgG levels varied according to age groups (from 4.3 ± 2.3 g/l in children under 6 months old to 11.4 ± 3.2 g/l in adolescents in the 10-<18 yo group). CG sensitivity and specificity to detect IgG below the reference range for all patients were 93.1% and 81.8%, respectively, and varied according to age group. Gamma sensitivity and specificity for all patients were 100% and 87.8%, respectively, and varied according to age group as well. We found serum IgG levels below the age reference level in 29 patients (2.4% of the cases) using CG or Gamma levels. Conclusion: Both CG and Gamma levels may be of utility as a screening tool for earlier diagnosis of antibody deficiency in children and adolescents under 18 yo.

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

Naturally occurring autologous anti-idiotypic antibodies. Participation in immune complex formation in selective IgA deficiency.

50% of individuals of selective IgA deficiency have high serum titers of antibody to bovine proteins, and high levels of circulating immune complexes that contain bovine antigens. Because in animal studies, immunization with antigen-antibody complexes is a very effective means of producing anti-idiotypic antibodies, we sought such autoantibodies in two sera known to have large amounts of anticasein. After IgG isolation and two-stage affinity chromatography, IgG-like material (molecular weights of H and L chains on SDS-PAGE), with binding activity for the F(ab')2 of anticasein were isolated from both sera. Pooled human gamma globulin or IgG myeloma proteins did not inhibit binding of specific anti-anticaseins to the corresponding anticasein, but sodium caseinate did block this binding (by 80 and 95%) indicating that most of these autoantibodies have affinity for the casein-binding site. Naturally occurring anti-idiotypic antibodies have been difficult to conclusively demonstrate in human sera; consequently, these experiments provide evidence of a unique model which may be used to explore the network theory of immunoglobulin regulation in humans.

Antibody response to the streptococcal group A-variant polysaccharide in BASILEA rabbits lacking kappa-polypeptide chains.

Rabbits from a variant strain called BASILEA, in which homozygotes express only lambda-type chains and heterozygotes have normal kappa/lambda ratios, were hyperimmunized with a streptococcal group A variant vaccine. Homozygotes (bas/bas) produced antibodies with lambda-chains, heterozygotes, however, produced predominantly antibodies with kappa-chains. The incidence of restricted high responders in the BASILEA strain was high; it was probably introduced by the original mutant rather than by the loss of kappa-chains (founder effect). The degree of heterogeneity of homozygotes is similar to the heterogeneity of heterozygotes, and to that of rabbits expressing kappa-chains. This suggests that in the rabbit, the repertoire of lambda-chain genes is of similar size to that of kappa-chain genes.

Human malignant T cells capable of inducing an immunoglobulin class switch.

Evidence is presented for the existence of a "switch" T cell derived from a patient with mycosis fungoides/Sezary's syndrome. The serum immunoglobulin profile in this patient revealed high IgG and IgA but no detectable IgM. Peripheral blood mononuclear cells from this patient secreted only IgG and IgA in the presence of pokeweed mitogen. T cells (Trac) co-cultured with normal allogeneic non-T cells and pokeweed mitogen resulted in only IgG and IgA PFC, with little or no IgM secretion. There was no evidence of active suppression of IgM. Rather, these T cells appeared to induce an Ig class switch from IgM to IgG and IgA, when co-cultured with mu+ tonsillar B cells. Further evidence was obtained using mononuclear cells derived from a patient with immunodeficiency and hyper-IgM, a syndrome characterized by a lack of IgG and IgA secretion. The addition of Trac cells to either peripheral blood mononuclear cells or non-T cells from a patient with hyper-IgM syndrome resulted in new secretion of IgG, with a concomitant decrease in IgM secretion, whereas control T cells were not effective in inducing secretion of any isotype other than IgM. Isolated Tac+ T cells from Trac appear to be responsible for this effect.

Clinical and immunological features in IgM deficiency.

BACKGROUND: IgM deficiency is a dysgammaglobulinemia characterized by isolated low levels of serum IgM. Patients with IgM deficiency may exhibit various clinical manifestations. However, IgM deficiency still remains to be explored with regard to diagnosis and treatment. METHODS: Through a retrospective chart review, we investigated the clinical and immunological features of 15 symptomatic adult IgM-deficient patients who were referred to our immunology clinics over a 4-year period. RESULTS: The patients were comprised of 6 males and 9 females, with a mean age of 57.2 years. On initial evaluation, 12 patients (80%) presented with susceptibility to infections, 5 (33%) had atopic manifestations such as asthma and allergic rhinitis, 3 (20%) had both infections and atopy, 4 patients (28%) had fibromyalgia-like symptoms, 3 (20%) had autoimmune manifestations, and 1 patient had lymphoma. The mean serum IgM level was 27.4 mg/dl (range 14-39). Impaired specific antibody response to pneumococcal antigens in 5 out of 11 studied patients (45%) appeared to be a notable association. Subtle abnormalities in IgG subclasses, lymphocyte subsets and in vitro proliferative lymphocyte responses were observed. Five patients who were treated with intravenous immunoglobulin responded very well. CONCLUSION: We propose that a thorough immunological evaluation including specific antibody responses be undertaken in patients with IgM deficiency. IgM-deficient patients who present with recurrent/severe infections may benefit from immunoglobulin treatment particularly in the presence of impaired pneumococcal antibody responses.

Immunoglobulin A Dysgammaglobulinemia Is Associated with Pediatric-Onset Obsessive-Compulsive Disorder.

Background: Inflammation and immune dysregulation have been implicated in the pathogenesis of pediatric-onset obsessive-compulsive disorder (OCD) and tic disorders such as Tourette syndrome (TS). Though few replicated studies have identified markers of immune dysfunction in this population, preliminary studies suggest that serum immunoglobulin A (IgA) concentrations may be abnormal in these children with these disorders. Methods: This observational retrospective cohort study, conducted using electronic health records (EHRs), identified 206 children with pediatric-onset OCD and 1024 adults diagnosed with OCD who also had testing for serum levels of IgA. IgA deficiency and serum IgA levels in pediatric OCD were compared with IgA levels from children diagnosed with autism spectrum disorders (ASD; n = 524), tic disorders (n = 157), attention-deficit/hyperactivity disorder (ADHD; n = 534), anxiety disorders (n = 1206), and celiac disease, a condition associated with IgA deficiency (n = 624). Results: Compared with ASD and anxiety disorder cohorts, the pediatric OCD cohort displayed a significantly higher likelihood of IgA deficiency (OR = 1.93; 95% CI = 1.18-3.16, and OR = 1.98; 95% CI = 1.28-3.06, respectively), though no difference was observed between pediatric OCD and TS cohorts. Furthermore, the pediatric OCD cohort displayed similar rates of IgA deficiency and serum IgA levels when compared with the celiac disease cohort. The pediatric OCD cohort also displayed the highest percentage of IgA deficiency (15%,) when compared with TS (14%), celiac disease (14%), ADHD (13%), ASD (8%), and anxiety disorder (8%) cohorts. When segregated by sex, boys with OCD displayed a significantly higher likelihood of IgA deficiency when compared with all comparison cohorts except for celiac disease and tic disorders; no significant difference in IgA deficiency was observed between female cohorts. Pediatric OCD subjects also displayed significantly lower adjusted serum IgA levels than the ASD and anxiety disorder cohorts. Adults with OCD were also significantly less likely than children with OCD to display IgA deficiency (OR = 2.71; 95% CI = 1.71-4.28). When compared with children with celiac disease, no significant difference in IgA levels or rates of IgA deficiency were observed in the pediatric OCD cohort. Conclusions: We provide further evidence of IgA abnormalities in pediatric-onset OCD. These results require further investigation to determine if these abnormalities impact the clinical course of OCD in children.

Pediatric selective IgM immunodeficiency.

OBJECTIVE: Limited information exists on features of pediatric Selective IgM immunodeficiency (SIgMID). Previously published pediatric cases and 2 new cases are reviewed. METHODS: English literature from PubMed and references from relevant articles were reviewed. Previously reported cases and 2 new cases from an allergy/immunology practice were analyzed. RESULTS: Forty-nine reported cases of SIgMID presented with respiratory infections (77.6%), gastrointestinal disease (16.3%), skin disease (12.2%), and meningitis (8.2%). Mean serum IgM level was 16.5+/-13.8 mg/dL. Two patients were identified with SIgMID among 6300 active pediatric patients (0.03%) presenting with asthma, vasomotor rhinitis, and recurrent respiratory infections. In the 51 cases reported, none developed lymphoproliferative disease nor evolved into panhypogammaglobulinemia; four fatalities were reported. CONCLUSIONS: The prevalence of SIgMID in our pediatric population was 0.03%. In general, respiratory infections are the common comorbid conditions. Death and autoimmune disease are uncommon complications of pediatric SIgMID.

Selective immunoglobulin M deficiency in an adult with Streptococcus pneumoniae sepsis and invasive aspergillosis.

Primary selective immunoglobulin (Ig) M deficiency usually presents early in life with recurrent or severe infections caused by encapsulated and gram-negative organisms. Primary selective IgM deficiency in adults is rare and is usually associated with autoimmune diseases or malignant neoplasm. We performed an extensive immunological analysis of innate and adaptive immunity in an adult patient with possible primary selective IgM deficiency who presented with life-threatening Streptococcus pneumoniae septic shock and invasive Aspergillus fumigatus infection. The patient had no evidence of autoimmune disease or malignant neoplasm. Serum IgG, IgA, and IgE were normal; however, serum IgM levels and specific antibody titers against all 14 pneumococcal polysaccharide serotypes were consistently low. Complement CH50, C3, C4, and neutrophil phagocytosis and oxidative burst were normal. Toll-like receptor expression on monocytes was also normal. Therefore, adult patients with serious life-threatening and unusual infections should be investigated for possible selective primary IgM deficiency.