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Background The International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) has recently proposed nomenclature in order to harmonize ANA indirect immunofluorescence (IIF) pattern reporting. ICAP distinguishes competent-level from expert-level patterns. A survey was organized to evaluate reporting, familiarity, and considered clinical value of ANA IIF patterns. Methods Two surveys were distributed by European Autoimmunity Standardization Initiative (EASI) working groups, the International Consensus on ANA Patterns (ICAP) and UK NEQAS to laboratory professionals and clinicians. Results 438 laboratory professionals and 248 clinicians from 67 countries responded. Except for dense fine speckled (DFS), the nuclear competent patterns were reported by > 85% of the laboratories. Except for rods and rings, the cytoplasmic competent patterns were reported by > 72% of laboratories. Cytoplasmic IIF staining was considered ANA positive by 55% of clinicians and 62% of laboratory professionals, with geographical and expertise-related differences. Quantification of fluorescence intensity was considered clinically relevant for nuclear patterns, but less so for cytoplasmic and mitotic patterns. Combining IIF with specific extractable nuclear antigens (ENA)/dsDNA antibody testing was considered most informative. Of the nuclear competent patterns, the centromere and homogeneous pattern obtained the highest scores for clinical relevance and the DFS pattern the lowest. Of the cytoplasmic patterns, the reticular/mitochondria-like pattern obtained the highest scores for clinical relevance and the polar/Golgi-like and rods and rings patterns the lowest. Conclusion This survey confirms that the major nuclear and cytoplasmic ANA IIF patterns are considered clinically important. There is no unanimity on classifying DFS, rods and rings and polar/Golgi-like as a competent pattern and on reporting cytoplasmic patterns as ANA IIF positive.

BACKGROUND: Sheffield NARCOS (National Adverse Reactions Advisory Service) investigates suspected perioperative anaesthetic reactions using serial tryptase, urinary methylhistamine and clinical information. Further recommendations for additional allergy clinic assessment are provided. OBJECTIVE: To establish a robustly measurable protocol for identifying mast cell mediator (MMR) release in this cohort. To compare these thresholds with previous suggested thresholds and algorithms. METHOD: A review of 3,455 NARCOS cases referred with a suspected peri-operative allergic reaction. Tryptase, Urinary methylhistamine (UMH) and clinical details were analysed. 1746 cases were graded using the Ring and Messmer scale. Reaction grade, tryptase and UMH changes were compared with statistical and graphical presentations appropriate to non-normally distributed measurements using Analyse-IT software. RESULTS: Sensitive strategies such as 3μg/l or 20% are measurable, translatable and would substantially increase detection of potentially relevant changes in tryptases. Adequate quality assurance for low level measurement is needed. An incremental threshold of 20% would identify potential MMR in an additional 14% of cases with peak tryptase (Tp) between 5 and 14 μg/L and a further 15% with Tp below 5 μg/L. Further work is required to establish the diagnostic performance characteristics of this more sensitive approach. UMH also identified up to 120 further cases of potential MMR in absence of tryptase increments. CONCLUSION AND CLINICAL RELEVANCE: Future studies should establish and compare the predictive performance characteristics of each strategy against clinical phenotypes. A single agreed definition of positive serial tryptases is needed to enable robust evaluation of diagnostic strategies. This could serve as a harmonised standard for comparative studies of case series from different centres. This article is protected by copyright. All rights reserved.

Summary 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.

Background Flucloxacillin is a narrow-spectrum, beta-lactamase–resistant penicillin. Type I (IgE-mediated) and type IV (T-cell–mediated) reactions are less frequently reported than with other penicillins. Objective To undertake a detailed clinical characterization of a cohort of patients referred with suspected flucloxacillin hypersensitivity. Methods We retrospectively analyzed demographic characteristics, presentation, investigation, and management of 108 patients presenting to 4 UK centers. Patients underwent skin prick and intradermal testing with flucloxacillin, major (benzylpenicilloyl poly- l -lysine) and minor determinants, amoxicillin, and benzylpenicillin with immediate and, where appropriate, delayed reading of the test. In the immediate group, a further 14 patients were tested to ampicillin and 16 to Augmentin (co-amoxiclav—combination of clavulanic acid and amoxicillin). Skin test–negative patients underwent oral drug provocation. A multistep protocol was used, depending on risk assessment. Results Forty of 108 (37%) patients were diagnosed with hypersensitivity to flucloxacillin, of whom 33 (82.5%) showed immediate and 7 (17.5%) nonimmediate hypersensitivity, respectively. In the immediate group, most reactions were severe: 19 of 33 (58%). Intradermal testing had a higher negative predictive value (86%) in the immediate group than in the nonimmediate group (67%). Only a minority of patients (6 of 17 [35%]) with IgE-mediated allergy were cross-sensitized on intradermal testing with other penicillins, compared with 3 of 4 (75%) in the delayed group. Conclusions Immediate hypersensitivity reactions to flucloxacillin are more common than delayed. Cross-sensitization to other penicillins appears higher in delayed reactions than in immediate. The negative predictive value of intradermal testing is higher in the immediate group than in the nonimmediate group. Drug provocation testing remains the diagnostic criterion standard.

Introduction Paediatric ESPGHAN guidelines support a diagnosis of coeliac disease (CD) when immunoglobulin-A anti-tissue transglutaminase (IgA tTG) antibody titres are >10 times the upper limit of normal (ULN) and combined with supportive criteria. This study examines whether serological testing alone could be sufficient for diagnosis in adult patients, thus avoiding the need for duodenal biopsies. Methods We performed a prospective analysis of CD patients diagnosed in a University hospital. Symptoms of CD, villous atrophy (VA) on biopsy, IgA-endomysial (IgA-EMA) antibodies, tTG and Human Leukocyte Antigen (HLA) genotype were used for analysis. We then compared the TTG antibody level against small bowel histology. Results 443 CD patients (66.8% female, median age 41 years, range 15–84 years) were diagnosed between 2008 and 2016. 56.9% (n=252, 95% CI=52.12–61.53) had a tTG value of greater than 10 times the ULN. 292 fulfilled ESPGHAN guidelines for features of malabsorption (diarrhoea=157, weight loss=45 and anaemia=190). Of these symptomatic patients, 70.4% (n=179, 95% CI=64.86–76.08) had a tTG value 10 x ULN. The proportion reaching the 10 x tTG threshold was 55.4% (n=87, 95% CI=47.64–63.19) for diarrhoea, 60.0% (n=27, 95% CI=45.69–74.31) for weight loss, and 74.2% (n=141, 95% CI=67.99–80.43) for anaemia. Of the 151 patients who did not experience malabsorptive features, 49.0% met the 10 x ULN tTG level (n=74, 95%CI=41.03–56.98). The sensitivity of tTG antibodies and EMA antibodies for predicting VA was 93.2% (95% CI=90.89–95.57) and 90.7% respectively (95% CI=88.05–93.44). Combined tTG and EMA was 98.6% (95% CI=97.67–99.72). All patients had compatible HLA typing, thereby failing to add any further diagnostic value. Conclusions 56.9% of patients would have been correctly diagnosed with CD and avoided a duodenal biopsy using an IgA tTG threshold of >10 times the ULN. Symptoms and HLA typing did not add any supportive information. This study provides evidence that a biopsy avoidance strategy may be implemented into adult gastroenterological practice.

Summary We describe an observational survey of diagnostic pathways in 104 patients attending four specialist allergy clinics in the United Kingdom following perioperative hypersensitivity reactions to chlorhexidine reactions. The majority were life-threatening. Men undergoing urological or cardiothoracic surgery predominated. Skin prick testing and specific immunoglobulin (sIg)E testing were the most common tests used for diagnosis. Fifty-three per cent of diagnoses were made on the basis of a single positive test. Where multiple tests were performed the sensitivity of intradermal, basophil activation and skin prick testing was 68% (50–86%), 50% (10–90%) and 35% (17–55%), respectively. Seven per cent were negative on screening tests initially, and 12 cases were only positive for a single test despite multiple testing. Intradermal tests appeared most sensitive in this context. Additional sensitization to other substances used perioperatively, particularly neuromuscular blocking agents (NMBA), was found in 28 patients, emphasizing the need to test for possible allergy to all drugs to which the patient was exposed even where chlorhexidine is positive.

Background In cerebrospinal fluid (CSF) spectrophotometry, if the net bilirubin absorbance (NBA) and net oxyhaemoglobin absorbance (NOA) are both raised with a visible oxyhaemoglobin peak, the revised national guidelines for analysis of CSF bilirubin advise interpreting the results as ‘Consistent with subarachnoid haemorrhage (SAH)’ regardless of the CSF total protein concentration of the specimen. We wanted to study the range of CSF total protein concentrations found in confirmed SAH to establish if the CSF total protein value can give further guidance on the likelihood of SAH. Methods Consecutive cases from five different hospital sites were included if the CSF NBA was greater than 0.007 AU and the NOA was greater than 0.02 AU with a visible oxyhaemoglobin peak. For the cases identified, the laboratory information management system and patient records were interrogated to identify the total protein concentration of the CSF specimen and whether SAH had ultimately been confirmed or excluded by other methods and supporting evidence. Results Results from 132 patients were included. The CSF total protein range in confirmed SAH was 0.23–3.08 g/L with a median concentration of 0.7 g/L (n = 51). In the SAH excluded group, the CSF total protein range was 0.43–29 g/L with a median concentration of 1.9 g/L (n = 81). Conclusions Although confirmed SAH was not associated with the very highest concentrations of CSF total protein, a definite CSF protein cut-off concentration above which SAH could reliably be excluded cannot be recommended.

A proportion of people living with common variable\ud immunodeficiency disorders develop granulomatouslymphocytic\ud interstitial lung disease (GLILD). We aimed to\ud develop a consensus statement on the definition, diagnosis, and\ud management of GLILD. All UK specialist centers were contacted\ud and relevant physicians were invited to take part in a 3-round\ud online Delphi process. Responses were graded as Strongly Agree,\ud Tend to Agree, Neither Agree nor Disagree, Tend to Disagree,\ud and Strongly Disagree, scored D1, D0.5, 0, L0.5, and L1,\ud respectively. Agreement was defined as greater than or equal to\ud 80% consensus. Scores are reported as mean – SD. There was\ud 100% agreement (score, 0.92 – 0.19) for the following\ud definition: “GLILD is a distinct clinico-radio-pathological ILD\ud occurring in patients with [common variable immunodeficiency\ud disorders], associated with a lymphocytic infiltrate and/or\ud granuloma in the lung, and in whom other conditions have been\ud considered and where possible excluded.” There was consensus\ud that the workup of suspected GLILD requires chest computed\ud tomography (CT) (0.98 – 0.01), lung function tests (eg, gas\ud transfer, 0.94 – 0.17), bronchoscopy to exclude infection (0.63 –\ud 0.50), and lung biopsy (0.58 – 0.40). There was no consensus on\ud whether expectant management following optimization of\ud immunoglobulin therapy was acceptable: 67% agreed, 25%\ud disagreed, score 0.38 – 0.59; 90% agreed that when treatment\ud was required, first-line treatment should be with corticosteroids\ud alone (score, 0.55 – 0.51).

BackgroundSheffield National Adverse Reactions Consultancy Service (NARCOS) investigates and triages suspected perioperative anaesthetic reactions to allergy clinics, using serial tryptase samples, urinary methylhistamine and clinical information on a pro forma request.ObjectiveTo determine if current UK guidelines on serial tryptase samples are achieved and describe the patterns of tryptase release.MethodA retrospective review of 3455 NARCOS cases. Tryptase and clinical details were analysed. 1746 had sufficient clinical information to grade the reactions according to the Ring and Messmer scale.Results98% provided two or more acute samples, but only 60% supplied clinical information. 308 patients never dropped within the reference range over three samples.ConclusionsGood compliance with UK guidelines for tryptase measurements is achievable in this long-term cohort, but obtaining clinical details at referral remains a challenge. Sample sequence labelling and apparent timing may be misleading. Baseline tryptase may frequently need to be rechecked in allergy clinics to estimate true peak-to-trough changes.

Summary Mast cell tryptase (MCT) is a key diagnostic test for mastocytosis and anaphylaxis. High serum tryptase levels are also one of the risk factors for adverse reaction in venom immunotherapy, yet occasional patients are seen with raised levels in the absence of either diagnosis. False positive results can be due to assay interference by heterophilic antibodies such as rheumatoid factor (RF) and human anti-mouse antibodies (HAMA). We therefore investigated heterophilic antibody interference by rheumatoid factor activity and HAMA as a cause of raised MCT results in the Phadia tryptase assay. Serum samples from 83 patients were assayed for MCT and rheumatoid factor before and after the use of heterophilic antibody blocking tubes (HBT). Samples with more than 17% reduction in MCT with detectable RF were then assayed for HAMA. Fourteen (17%) of the 83 samples with positive RF showed a >17% decrease in mast cell tryptase after HBT blocking. Post-HBT, eight of 14 (57%) reverted from elevated to normal range values with falls of up to 98%. RF levels were also decreased significantly (up to 75%). Only one of the 83 tested was apparently affected by HAMA in the absence of detectable IgM RF. In conclusion, any suspicious MCT result should be checked for heterophilic antibodies to evaluate possible interference. False positive MCT levels can be caused by rheumatoid factor. We suggest a strategy for identifying assay interference, and show that it is essential to incorporate this caveat into guidance for interpretation of MCT results.

Cryoglobulins are serum immunoglobulins that precipitate at temperatures below 37°C and re-dissolve on warming. Cryoglobulinaemia leads to variable symptoms including characteristic purpura, ischaemia of extremities, renal failure, peripheral neuropathy, abdominal pain secondary to intestinal ischaemia and arthralgias. Cryoglobulin testing is underutilized in clinical practice. It has been neglected in clinical laboratories and by clinicians due to several factors, such as the length of time it takes for serum cryoglobulin analysis to be performed in the laboratory, the perceived difficulty in getting optimal sampling conditions and a failure to appreciate that even apparently low levels of cryoglobulin can be associated with severe symptoms in some patients. The most important variable confounding standardization of cryoglobulin testing is improper sample handling. A recent report critically appraising the current practice of cryoglobulin evaluation in 137 laboratories in Europe by United Kingdom National External Quality Assurance Scheme (UKNEQAS) illustrated the wide variability in practice. Although many clinical laboratories perform cryoglobulin evaluation, there are widespread differences in the methodology used and the care with which this is carried out and this leads to considerable intralaboratory and interlaboratory variability. The most common sources of error are false-negative results due to loss of cryoprecipitate during transport and storage. Better standardization is needed to avoid missed diagnoses and improve the comparability of results. Laboratories should ensure that sample temperature is maintained at 37°C until the serum is separated. In this article, we briefly review the classification and clinical features of cryoglobulins and suggest best practice guidelines for laboratory detection and identification of cryoglobulins.

Background & Aims: The optimal serologic tests for the detection of celiac disease and follow-up assessment remains controversial. Our aim was to evaluate all current immunologic assays for diagnosing celiac disease using the gold standard of duodenal biopsy. We also assessed whether tissue transglutaminase (tTG) antibody is a quantitative marker for histologic severity. Methods: Consecutive adult patients referred for gastroscopy without a previous known diagnosis of celiac disease were recruited (group 1). Concurrently, patients with a known diagnosis of celiac disease on a gluten-free diet for more than 1 year undergoing repeat duodenal biopsy were identified (group 2). All patients had duodenal biopsies and serologic analysis performed for immunoglobulin(Ig) A and antibodies to human immunoglobulin (Ig)A-tTG, IgA-gliadin, IgG-gliadin, and IgA-endomysial antibody. Results: Two thousand patients were recruited in the first group. Seventy-seven (3.9%) patients were diagnosed with new celiac disease. The sensitivity, specificity, positive predictive value, and negative predictive value for IgA tTG were 90.9%, 90.9%, 28.6%, and 99.6%. When adopting a 2-step approach using tTG first and then EMA the sensitivity, specificity, positive predictive value, and negative predictive value was 85.7%, 98.6%, 71.7%, and 99.7%, respectively. The use of nondeamidated IgA/IgG gliadin antibodies conferred no additional diagnostic benefit when considering the detection of adult celiac disease. In the second group 48 patients with celiac disease on a gluten-free diet were identified. Sixteen of 48 of these patients had persisting villous atrophy, but 7 of 16 (44%) had a normal tTG level. Conclusions: IgA tTG alone is a sensitive marker for celiac disease. A normal tTG level does not predict recovery of villous atrophy in patients with celiac disease on a gluten-free diet.

The updated ESPGHAN guidance on coeliac disease recommends the use of common multiples of the upper limit of normal (ULN) for IgA tissue transglutaminase antibodies (TG2) when deciding which diagnostic pathway to follow. The current lack of standardisation between assays makes it difficult to harmonise results between centres as different performance characteristics are observed with each assay. This variability is shown in data from external quality assessment distributions. As a result, the updated guidance is too generalised for use with all the commercial TG2 kits and is therefore not translatable for use in all centres.

This case report highlights the immunological considerations in patients who are diagnosed with disseminated infection due to BCG or environmental mycobacteria.

Results The patient (smoker) complained on a variety of symptoms that were bothering her for more than 3 years: headaches, significant fatigue, nasal blockage, breathlessness on exercise and occasionally at rest. There were no ocular symptoms. She had several courses of antibiotics during this period with slight improvement. Careful history revealed progressive deterioration in her condition; there was no difference in her symptom pattern through the season although she noted that she had more sinus pains in winter. There was a dog, but no dampness in the house. There was no personal or family history of atopy. Anterior rhinoscopy had appearance of acute inflammation with no structural abnormalities. Spirometry showed FEV1 95%, FVC 92% and Peak flow 85% predicted. Skin prick test with valid controls, was negative to a standard panel of aeroallergens including dog. Immunocap ISAC test was offered to broaden the spectrum of allergens tested. This showed mono-sensitisation to Dog rCan f 5 Arginine esterase 1,5 ISU and negative to all other components.

Aims Immunoglobulin A (IgA) measurement is advocated when case finding for coeliac disease in patients with Type 1 diabetes mellitus. Currently, there is a paucity of contemporary studies assessing IgA deficiency in Type 1 diabetes. This study evaluates the prevalence of IgA deficiency in individuals with Type 1 diabetes, compared with patients with coeliac disease and control subjects. In addition, we evaluate whether routine IgA measurement is justifiable when case finding for coeliac disease in patients with Type 1 diabetes. Methods All patients were assessed using IgA endomysial antibodies, IgA anti-tissue transglutaminase antibodies and total IgA levels. Altogether, 2434 individuals were tested: 1000 patients with Type 1 diabetes, 234 patients with coeliac disease and 1200 population control subjects. Definitive IgA deficiency was defined as total IgA levels

Summary National Institute of Clinical Excellence (NICE) and European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidance for the diagnosis of coeliac disease has been published. However, there is some controversy regarding the advice on the use of stratifying levels of immunoglobulin (IgA) tissue transglutaminase antibody (TG2) test positivity in the absence of test standardization and the vagueness of the indication to test equivocal samples. Using repeat service audit, we demonstrate that a combination of TG2 followed by IgA endomysial antibodies (EMA) is the best strategy for all degrees of mucosal abnormality using our test combination. Reliance upon immunoassay titre is not as effective, and cannot be applied consistently across populations in the absence of assay standardization. Guidelines advocating the use of tests should involve experts in laboratory diagnostics and external quality assurance to ensure that errors of generalization do not occur and that test performance is achievable in routine diagnostic use.

Objective To determine the prevalence, prognostic value, and response to thyroxine therapy of thyroid peroxidase antibody (TPOAb) in women with unexplained recurrent miscarriage (RM). Design Observational, cohort study. The index cases included women with unexplained RM who tested positive for thyroid peroxidase antibody, and control cases included women with unexplained RM who tested negative for the antibodies; a second age-matched control group included women with RM who had a known cause for the repeated pregnancy loss. Setting Tertiary referral center for RM. Patient(s) A total of 496 women with unexplained RM and 220 women with known diagnoses of RM who had a TPOAb test. Intervention(s) Thyroxine replacement (50 μg daily during pregnancy) was begun in some patients who tested positive for thyroid peroxidase antibody, irrespective of TSH level. Main Outcome Measure(s) Miscarriage and live birth rates of a subsequent pregnancy. Result(s) A total of 496 women with unexplained RM who had a TPOAb test were included in the study. Of these, 10.7% of subjects tested positive for TPOAb. The prevalence of TPOAb in control subjects who had a known cause for RM was 11.8%. The live birth rate of the first pregnancies after referral was 64%, 53%, and 58% in TPOAb-negative, TPOAb-positive with thyroxine treatment, and TPOAb-positive without treatment subjects; there was no significant difference in the outcome between any two or three groups, or between those who tested positive or negative for TPOAb. Among women who tested positive for TPOAb, there was no difference in the antibody titer between women with unexplained RM and those with a known cause for the pregnancy loss. Women who tested positive for TPOAb were significantly more likely to have TSH levels above the normal range (≥4.2 mIU/L). Conclusion(s) The prevalence of TPOAb-positive results in women with unexplained RM is not higher than in the general population, TPOAb-positive status does not have a prognostic value regarding the outcome of a subsequent pregnancy, and empirical thyroxine therapy in those who tested positive did not seem to improve outcome.

To assess current practice in the detection, analysis, and reporting of cryoglobulins, a questionnaire was sent to 140 laboratories. Only 36% of laboratories used standard procedures (tube preheating, transport in container, and sedimentation and/or centrifugation at 37 °C) to ensure that the temperature did not drop below 37 °C until after serum separation. Time periods allowed for cryoprecipitation at 4 °C varied from 12 h to 9 days, with 30% of laboratories allowing precipitation for

To underline the importance of correctly examining cerebrospinal fluid, we describe two patients with subarachnoid haemorrhage for whom cranial computerised tomography was normal and on whom lumbar puncture was subsequently done. Both had clear and colourless cerebrospinal fluid, but subsequent spectrophotometry showed bilirubin. Cerebral angiography then showed a ruptured aneurysm in each patient. Case 1 A 28 year old woman presented with nausea and diplopia. She had global headache which had awoken her 14 days before. This was associated with neck pain and stiffness, vomiting for two days, and photophobia for five to six days. On examination, severe bilateral papilloedema, and signs of a VIth nerve palsy were present. Cranial computerised …

Editor,—We report a case of bilateral granulomatous uveitis, which prompted extensive diagnostic review in a 20 year old woman with a long history of recurrent infection and idiopathic thrombocytopenia. Investigations allowed the definitive diagnosis of common variable immunodeficiency with granulomas (granulomatous antibody deficiency syndrome.) To our knowledge this is the first reported case of granulomatous uveitis in association with granulomatous antibody deficiency (GAD). We discuss the features of GAD, and how it may be distinguished from sarcoidosis. ### CASE REPORT A 20 year old woman presented with sudden onset blurred vision. Examination revealed a bilateral granulomatous uveitis with mutton-fat keratic precipitates and anterior chamber cells. Two weeks later she developed bilateral optic disc swelling with multifocal areas of choroidal pallor in her left eye (Fig 1). There was no vitritis …