Quantification of human C1 esterase inhibitor protein using an automated turbidimetric immunoassay.

BACKGROUND: Impaired levels or function of C1 inhibitor (C1-INH) results in angioedema due to increased bradykinin. It is important to distinguish between angioedema related to C1-INH deficiency and that caused by other mechanisms, as treatment options are different. In hereditary (HAE) and acquired (AAE) angioedema, C1-INH concentration is measured to aid patient diagnosis. Here, we describe an automated turbidimetric assay to measure C1-INH concentration on the Optilite® analyzer. METHODS: Linearity, precision, and interference were established over a range of C1-INH concentrations. The 95th percentile reference interval was generated from 120 healthy adult donors. To compare the Optilite C1-INH assay with a predicate assay used in a clinical laboratory, samples sent for C1-INH investigation were used. The predicate results were provided to allow comparison. RESULTS: The Optilite C1-INH assay was linear across the measuring range at the standard sample dilution. Intra and interassay variability was <6%. The 95th percentile adult reference interval for the assay was 0.21-0.38 g/L. There was a strong correlation between the Optilite concentrations and those generated with the predicate assay (R2  = 0.94, P < 0.0001, slope y = 0.83x). All patients with Type I HAE (n = 24) and AAE (n = 3) tested had concentrations below the measuring range in both assays, while all patients with unspecified angioedema (UAE), not diagnosed with HAE or AAE had values within the reference range. CONCLUSION: The Optilite assay allows the automated and precise quantification of C1-INH concentrations in patient samples. It could therefore be used as a tool to aid the investigation of patients with angioedema.

Clozapine is associated with secondary antibody deficiency.

BACKGROUND: Schizophrenia affects 1% of the population. Clozapine is the only medication licensed for treatment-resistant schizophrenia and is intensively monitored to prevent harm from neutropenia. Clozapine is also associated with increased risk of pneumonia although the mechanism is poorly understood.AimsTo investigate the potential association between clozapine and antibody deficiency. METHODS: Patients taking clozapine and patients who were clozapine-naive and receiving alternative antipsychotics were recruited and completed a lifestyle, medication and infection-burden questionnaire. Serum total immunoglobulins (immunoglobulin (Ig)G, IgA, IgM) and specific IgG antibodies to haemophilus influenzae type B, tetanus and IgG, IgA and IgM to pneumococcus were measured. RESULTS: Immunoglobulins were all significantly reduced in the clozapine-treated group (n = 123) compared with the clozapine-naive group (n = 111). Odds ratios (ORs) for a reduction in clozapine:control immunoglobulin values below the fifth percentile were IgG, OR = 6.00 (95% CI 1.31-27.44); IgA, OR = 16.75 (95% CI 2.18-128.60); and IgM, OR = 3.26 (95% CI 1.75-6.08). These findings remained significant despite exclusion of other potential causes of hypogammaglobulinaemia. In addition, duration on clozapine was associated with decline in IgG. A higher proportion of the clozapine-treated group reported taking more than five courses of antibiotics in the preceding year (5.3% (n = 5) versus 1% (n = 1). CONCLUSIONS: Clozapine use was associated with significantly reduced immunoglobulin levels and an increased proportion of patients using more than five antibiotic courses in a year. Antibody testing is not included in existing clozapine monitoring programmes but may represent a mechanistic explanation and modifiable risk factor for the increased rates of pneumonia and sepsis-related mortality previously reported in this vulnerable cohort.Declaration of interestS.J. has received support from CSL Behring, Shire, LFB, Biotest, Binding Site, Sanofi, GSK, UCB Pharma, Grifols, BPL SOBI, Weatherden, Zarodex and Octapharma for projects, advisory boards, meetings, studies, speaker and clinical trials.

Increased Respiratory Viral Detection and Symptom Burden Among Patients with Primary Antibody Deficiency: Results from the BIPAD Study.

BACKGROUND: Patients with primary antibody deficiency (PAD) are at increased risk of respiratory tract infections, but our understanding of their nature and consequences remains limited. OBJECTIVE: To define the symptomatic and microbial burden of upper airway infection in adults with PAD relative to age-matched controls. METHODS: Prospective 12-month observational study consisting of a daily upper and lower airway symptom score alongside fortnightly nasal swab with molecular detection of 19 pathogen targets. RESULTS: A total of 44 patients and 42 controls (including 34 household pairs) were recruited, providing more than 22,500 days of symptom scores and 1,496 nasal swabs. Swab and questionnaire compliance exceeded 70%. At enrollment, 64% of patients received prophylactic antibiotics, with a 34% prevalence of bronchiectasis. On average, patients with PAD experienced symptomatic respiratory exacerbations every 6 days compared with 6 weeks for controls, associated with significant impairment of respiratory-specific quality-of-life scores. Viral detections were associated with worsening of symptom scores from a participant's baseline. Patients with PAD had increased odds ratio (OR) for pathogen detection, particularly viral (OR, 2.73; 95% CI, 2.09-3.57), specifically human rhinovirus (OR, 3.60; 95% CI, 2.53-5.13) and parainfluenza (OR, 3.06; 95% CI, 1.25-7.50). Haemophilus influenzae and Streptococcus pneumoniae were also more frequent in PAD. Young child exposure, IgM deficiency, and presence of bronchiectasis were independent risk factors for viral detection. Prophylactic antibiotic use was associated with a lower risk of bacterial detection by PCR. CONCLUSIONS: Patients with PAD have a significant respiratory symptom burden associated with increased viral infection frequency despite immunoglobulin replacement and prophylactic antibiotic use. This highlights a clear need for future therapeutic trials in the population with PAD, and informs future study design.

Development of a high-throughput SARS-CoV-2 antibody testing pathway using dried blood spot specimens.

BACKGROUND: Serological assays for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have roles in seroepidemiology, convalescent plasma-testing, antibody durability and vaccine studies. Currently, SARS-CoV-2 serology is performed using serum/plasma collected by venepuncture. Dried blood spot (DBS) testing offers significant advantages as it is minimally invasive, avoids venepuncture with specimens being mailed to the laboratory. METHODS: A pathway utilizing a newborn screening laboratory infrastructure was developed using an enzyme-linked immunosorbent assay to detect IgG antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein in DBS specimens. Paired plasma and DBS specimens from SARS-CoV-2 antibody-positive and -negative subjects and polymerase chain reaction positive subjects were tested. DBS specimen stability, effect of blood volume and punch location were also evaluated. RESULTS: DBS specimens from antibody-negative (n = 85) and -positive (n = 35) subjects and polymerase chain reaction positive subjects (n = 11) had a mean (SD; range) optical density (OD) of 0.14 (0.046; 0.03-0.27), 0.98 (0.41; 0.31-1.64) and 1.12 (0.37; 0.49-1.54), respectively. An action value OD >0.28 correctly assigned all cases. The weighted Deming regression for comparison of the DBS and the plasma assay yielded: y = 0.004041 + 1.005x, r = 0.991, Sy/x 0.171, n = 82. Extraction efficiency of antibodies from DBS specimens was >99%. DBS specimens were stable for at least 28 days at ambient room temperature and humidity. CONCLUSIONS: SARS-CoV-2 IgG receptor-binding domain antibodies can be reliably detected in DBS specimens. DBS serological testing offers lower costs than either point of care or serum/plasma assays that require patient travel, phlebotomy and hospital/clinic resources; the development of a DBS assay may be particularly important for resource poor settings.

Clinical and laboratory characteristics of clozapine-treated patients with schizophrenia referred to a national immunodeficiency clinic reveals a B-cell signature resembling common variable immunodeficiency (CVID).

AIMS: An association between antibody deficiency and clozapine use in individuals with schizophrenia has recently been reported. We hypothesised that if clozapine-associated hypogammaglobulinaemia was clinically relevant this would manifest in referral patterns. METHODS: Retrospective case note review of patients referred and assessed by Immunology Centre for Wales (ICW) between January 2005 and July 2018 with extraction of clinical and immunological features for individuals with diagnosis of schizophrenia-like illness. RESULTS: 1791 adult patients were assessed at ICW during this period; 23 patients had a psychiatric diagnosis of schizophrenia or schizoaffective disorder. Principal indications for referral were findings of low calculated globulin and immunoglobulins. Clozapine was the single most commonly prescribed antipsychotic (17/23), disproportionately increased relative to reported use in the general schizophrenia population (OR 6.48, 95% CI: 1.79 to 23.5). Clozapine therapy was noted in 6/7 (86%) of patients subsequently requiring immunoglobulin replacement therapy (IgRT). Marked reduction of class-switched memory B cells (CSMB) and plasmablasts were observed in clozapine-treated individuals relative to healthy age-matched controls. Clozapine duration is associated with CSMB decline. One patient discontinued clozapine, with gradual recovery of IgG levels without use of IgRT. CONCLUSIONS: Our findings are consistent with enrichment of clozapine-treatment within schizophrenic individuals referred for ICW assessment over the last 13 years. These individuals displayed clinical patterns closely resembling the primary immunodeficiency common variable immunodeficiency, however appears reversible on drug cessation. This has diagnostic, monitoring and treatment implications for psychiatry and immunology teams and directs prospective studies to address causality and the wider implications for this patient group.

Proteinase-activated receptor-2 modulates human macrophage differentiation and effector function.

Proteinase-activated receptor-2 (PAR-2) was shown to influence immune regulation; however, its role in human macrophage subset development and function has not been addressed. Here, PAR-2 expression and activation was investigated on granulocyte macrophage (GM)-CSF(M1) and macrophage (M)-CSF(M2) macrophages. In both macrophages, the PAR-2-activating peptide, SLIGKV, increased PAR-2 expression and regulated TNF-α and IL-10 secretion in a manner similar to LPS. In addition, HLA-DR on M1 cells also increased. Monocytes matured to an M1 phenotype in the presence of SLIGKV had reduced cell area, and released less TNF-α after LPS challenge compared with vehicle (P < 0.05, n = 3). Cells matured to an M2 phenotype with SLIGKV also had a reduced cell area and made significantly more TNF-α after LPS exposure compared to vehicle (P < 0.05, n = 3) with reduced IL-10 secretion (P < 0.05, n = 3). Thus, PAR-2 activation on macrophage subsets regulates HLA-DR and PAR-2 surface expression, and drives cytokine production. In contrast, PAR-2 activation during M1 or M2 maturation induces altered cell morphology and skewing of phenotype, as evidenced by cytokine secretion. These data suggest a complex role for PAR-2 in macrophage biology and may have implications for macrophage-driven disease in which proteinase-rich environments can influence the immune process directly.