Paediatric reference ranges for faecal calprotectin: a UK study.

Background Faecal calprotectin has been shown to be a useful non-invasive marker for the diagnosis and monitoring of inflammatory bowel disease in children and adults. Although there are well-established reference ranges for the diagnosis of inflammatory bowel disease in adults, these have been less well studied in children. The objective was to establish reference ranges in our local population. Method All faecal calprotectin results from 2011 to 2014 were retrospectively collated and grouped according to patient age. Probability plots were used to determine expected upper limit of normal for each age group, and Mann-Whitney test was used to determine statistical difference between groups. Results Upper limit of normal for age groups 1-3.9 years, 4-17.9 years and 18 years plus were 77, 62 and 61 µg/g, respectively. There was a significant difference ( P = 0.0013) between the median calprotectin concentration for the age group 1-3.9 years ( n = 87) and 4-17.9 years ( n = 636) and between the age group 1-3.9 years and 18 years plus ( n = 7953, P = 0.0001), but there was no significant difference between the age groups 4-17.9 years and 18 years plus ( P = 0.4206). Conclusions In our local population, faecal calprotectin varies with age. Children aged 1-3.9 years had higher concentrations of faecal calprotectin than adults, but there was no significant difference in faecal calprotectin between older children and adults. This is in agreement with other published studies; however, the faecal calprotectin upper limit of normal calculated for children aged 1-3.9 years was lower than has been observed elsewhere.

Uncertainty of measurement: an immunology laboratory perspective.

'Measurement uncertainty of measured quantity values' (ISO15189) requires that the laboratory shall determine the measurement uncertainty for procedures used to report measured quantity values on patients' samples. Where we have numeric data measurement uncertainty can be expressed as the standard deviation or as the co-efficient of variation. However, in immunology many of the assays are reported either as semi-quantitative (i.e. an antibody titre) or qualitative (positive or negative) results. In the latter context, measuring uncertainty is considerably more difficult. There are, however, strategies which can allow us to minimise uncertainty. A number of parameters can contribute to making measurements uncertain. These include bias, precision, standard uncertainty (expressed as standard deviation or coefficient of variation), sensitivity, specificity, repeatability, reproducibility and verification. Closely linked to these are traceability and standardisation. In this article we explore the challenges presented to immunology with regard to measurement uncertainty. Many of these challenges apply equally to other disciplines working with qualitative or semi-quantitative data.

Food allergy testing.

Food allergy (IgE-mediated hypersensitivity) is a common clinical problem affecting approximately 15% of children in the Western world. These hypersensitivity reactions tend to be "immediate" (typically within minutes of food exposure), and clinical features may range from mild to life threatening (anaphylaxis). Detailed clinical history is critical to correct diagnosis. Available laboratory tests have limitations not least poor positive predictive value and limited repertoire. Laboratory tests should support clinical diagno sis not vice versa.

A simple immunofixation test for induced C3 degradation in disease states (including C3 nephritic factor) and its correlation with kidney biopsy.

Complement dysregulation from an uncontrolled activation of the alternate pathway can be mediated by C3 Nephritic Factor and results in C3 glomerulopathy. Identification of C3 degradation products C3c and C3d in patient serum provides evidence of uncontrolled complement activation. It is possible to detect C3c and C3d in patient serum by an immunofixation assay which induces in vitro C3 degradation. The clinical performance of the immunofixation assay has been assessed by comparing the assay results with findings from immunostaining of kidney biopsies. The immunofixation assay is a simple and reliable technique for detection of C3 degradation on a widely available platform and can be used to provide corroborative evidence of acquired complement dysregulation in patients with C3 glomerulopathy.

Abnormalities of serum-free light chain in patients with primary antibody deficiency in the absence of B lymphocyte clonality.

AIMS: A review of practice to determine whether serum-free light chain (SFLC) assays are helpful in detecting underlying clonal B-cell disorders or amyloidosis in patients with primary antibody deficiency (PAD) and recurrent infection. METHODS: SFLC were assayed by nephelometry (BN2 nephelometer, Siemens; FREELITE assay, Binding Site). We reviewed SFLC test results recorded in our regional laboratory over a 4-year time period; 20 adults with PAD were identified as having been tested on at least two occasions. RESULTS: Of 20 patients, 4 with PAD had abnormal serum-free kappa/lambda (K/L) ratios but no evidence of B-cell clonality. We also found extremely low levels of kappa and or lambda (below the limits of reliable detection) in 19/20 PAD cases (mostly common variable immunodeficiency), such that in many, ratios were not calculable. CONCLUSIONS: The data suggest that the abnormal ratios are generated by an inability to produce and/or secrete SFLCs, particularly kappa FLC. In this small initial study, we seek to highlight PAD cases where a suspicious K/L ratio, typically with very low absolute quantities of SFLCs, most likely points to B-cell dysfunction, rather than to B lymphocyte clonality.

Cinryze (C1-inhibitor) for the treatment of hereditary angioedema.

Cinryze is a pasteurized, nanofiltered plasma derived concentrate of C1-inhibitor (pdC1-INH) licensed for the prophylactic treatment of hereditary angioedema. In a double-blind placebo-controlled crossover trial to evaluate Cinryze as prophylaxis, the frequency of attacks was halved (6.26 per 12 weeks on Cinryze versus 12.73 per 12 weeks on placebo). Furthermore, attacks were generally milder and of shorter duration. For treatment of acute attacks in patients receiving Cinryze, 1000 units, within 4 h of the start of an attack, the estimated time to the onset of unequivocal relief was reduced to 2 h, compared with more than 4 h in those treated with placebo. Cinryze and other similar products are going to change the future management of hereditary angioedema and have potential in other areas of medicine.

Immunoglobulin E deficiency: a forgotten clue pointing to possible immunodeficiency?

BACKGROUND: Patients with primary antibody deficiency often have delayed diagnosis. Very low IgE, found during investigations for allergy, may be a marker for other immunodeficiency. METHODS: We introduced a new laboratory policy of testing cases with very low IgE levels for possible linked antibody deficiency. The data represent an audit of routine results collected over two years. RESULTS: Very low IgE (≤2 IU/mL) was identified in 85/2622 (3.2%) routine patient samples. Two children and four adult patients were found to have one or more classes of immunoglobulin below the reference range for age. In 2/6, the initiative of the laboratory led to a new unsuspected diagnosis of antibody immunodeficiency. CONCLUSIONS: Common variable immunodeficiency continues to be overlooked as a primary cause of lung disease in adults. Very low serum IgE should trigger appropriate investigation (immunoglobulin quantification and serum electrophoresis).

Food allergy: which tests are worth doing and which are not?

Adverse reactions to foods may arise by a variety of mechanisms, both immune (IgE and non-IgE) and non-immune mediated. This article considers those assays useful in the diagnosis of Type 1 hypersensitivity to foods (IgE-based) and, importantly, discusses those assays where evidence is lacking for their use. In all cases of suspected food allergy, a full clinical history is indispensable in facilitating diagnosis. Total serum IgE is not a suitable screen for food allergy. Suspect allergens may be confirmed by either skin prick testing or serological assays for specific IgE. Several studies suggest concentrations of food-specific IgE at which there is a high probability of reaction on food challenge. These cut-off levels are now being used by physicians to direct clinical advice. However, it is important to note that not all studies agree on these limits and the chosen cut-off is dependent on the population studied and the assay used.

Proteomic analysis of the venom of Heterometrus longimanus (Asian black scorpion).

Venoms have evolved over millions of years into potent cocktails of bioactive peptides and proteins. These compounds can be of great value to the pharmaceutical industry for numerous clinical applications. In this study, a novel proteomic - bioinformatic approach was utilised, where chromatography followed by gel electrophoresis was utilised to separate the venom peptides/proteins of Heterometrus longimanus (Asian black scorpion). Purified peptides were analysed by tandem mass spectrometry, de novo sequenced and then homology matched against known peptides in the Swiss-Prot protein database. Numerous potentially biologically active peptide matches were discovered, and a simple scoring system applied to putatively assign functions to the peptides. As a validation of this approach, the functional composition of the experimentally derived proteome is similar to that of other scorpions, and contains a potent mix of toxins, antimicrobials and ionic channel inhibitors.

Opercular epithelial cells: a simple approach for in vitro studies of cellular responses in fish.

This study evaluated the efficacy of fish opercular external (skin) and inner (opercular membrane) epithelium as an in vitro model for toxic and other substances studies. The rainbow trout (Oncorhynchus mykiss) operculum was cultured in 12-well dishes containing sterile Leibovitz 15 (L-15) supplemented with glutamine medium during 24h at 9 degrees C, and the effect of copper, a toxic agent, and/or cortisol, an endogenous agent, on the epithelial cells was analyzed using light microscopy techniques. The opercula were submitted to four treatments: (i) control (Cont), L-15 medium only, (ii) 0.28 microM cortisol (Cort), (iii) 100 microM CuSO(4) (Cu), and (iv) 0.28 microM cortisol+100 microM CuSO(4) (Cort-Cu). The tissue condition after 24h incubation was analyzed by staining the mucous cells for neutral and acid mucosubstances. Cellular necrosis was evaluated by measuring the lactate dehydrogenase (LDH) leakage at 12 and 24h incubation. Cellular proliferation, apoptosis, metallothionein (MT) and glucocorticoid receptor (GR) expression were evaluated by immunohistochemistry. The LDH leakage was higher and the proliferating cell nuclear antigen (PCNA) positive-stained cells were lower in Cu treatment in both, epidermis and opercular membrane. Apoptotic cells in the opercular membrane were higher in the Cort and Cort-Cu treatments while, in the epidermis, they were higher in Cu and Cort-Cu treatments. GR-positive stained cells decreased significantly in all treatments in both epithelia and the MT-positive cells increased in the Cu and Cort-Cu treated groups. Copper showed to be a potent toxic stressor killing the cells via necrosis, decreasing the number of PCNA-positive cells and inducing MT synthesis while cortisol did not affect the MT synthesis, although it might stimulate apoptosis. The results are evidence that the opercular epithelia serve as a suitable model for studying in vitro effects of toxic agents, as well as endogenous factors on the cellular responses without interference of the physiological state of fish being useful to predict in vivo toxicity.

Cerebellar ataxia, peripheral neuropathy, "gluten sensitivity" and anti-neuronal autoantibodies.

"Gluten sensitive" neurological syndromes (ataxia, peripheral neuropathy, and other conditions) have been hypothesised in patients with various idiopathic neuropathologies, detectable anti-gliadin antibodies and HLA-DQ2 or DQ7. Further investigation of these cases has suggested a high incidence of anti-neuronal antibodies (anti-Purkinje, anti- neuronal nuclear, anti-GAD). This study investigates this contentious area. Over a two-year period, from a local UK population base of two million, seeing over 5000 general neurology referrals per year, we collected 20 cases with idiopathic ataxia, and 32 with idiopathic peripheral neuropathy, and referred them all for blinded antibody testing. 30 adult healthy blood donors, and 7 cases of hereditary ataxia were used as control subjects. Anti-gliadin antibodies (IgG and or IgA) were found in 40% of cases with idiopathic ataxia, 34% with idiopathic peripheral neuropathy, 17% healthy blood donors and 43% with hereditary ataxia. None was positive for antiPurkinje cell or anti-neuronal nuclear antibodies. Only two patients with idiopathic ataxia were positive for antiGAD antibodies (one also being anti-gliadin positive). We were unable to confirm the findings of other groups. First, cases of so-called "gluten sensitive" neurological syndromes were extremely rare in our centre. Second, our idiopathic cases, whether they be gliadin antibody seropositive or not (i.e. "gluten sensitive" or not) were rarely neuronal autoantibody positive.

Celiac disease with mild to moderate histologic changes is a common cause of chronic diarrhea in Indian children.

OBJECTIVES: In developed countries, small bowel histology in coeliac disease is a spectrum, ranging from normal with increased intraepithelial lymphocytes to the classic flat mucosa. In developing countries, mild to moderate enteropathies in children with chronic diarrhea and growth failure are assumed to be caused by tropical sprue, persistent infections, or malnutrition with bacterial overgrowth. We report the prevalence and histology of coeliac disease in children with chronic diarrhea at a tertiary referral hospital in North India. METHODS: Two hundred fifty-nine children with symptoms indicating coeliac disease attended the All India Institute of Medical Sciences. Histology was graded after a modified Marsh classification. Serum immunoglobulin A anti-endomysial antibodies (AEA) were assayed using indirect immunofluorescence. Subjects with abnormal histology and positive AEA were put on a gluten free diet (GFD). Coeliac disease was diagnosed on small intestinal biopsy changes and a clinical response to a GFD. RESULTS: Severe enteropathies were present in 63 (24%) subjects, and 58 (92%) responded to a GFD. Sixty-six (25%) had moderate histologic changes, 61 responding to a GFD. AEA was positive in 56 of 63 patients with severe and 65 of 66 with moderate enteropathies. Fifty-seven children had mild enteropathies, and 19 of 20 with positive AEA responded clinically to a GFD. CONCLUSIONS: Coeliac disease is more common than previously believed. It presents a variable histology, and diagnoses may be missed or delayed if based only on severe enteropathies. Serology is a useful adjunct to diagnosis, and diagnostic criteria need to be developed appropriately for coeliac disease in developing countries despite limited facilities.

Dysgammaglobulinaemia in the elderly--a review and case studies.

An understanding of the possible causes of dysgammaglobulinaemia in the elderly helps to direct further investigation to establish a diagnosis. In this review we provide brief case studies to illustrate some of the disorders associated with dysgammaglobulinaemia in the elderly. We consider both hypergammaglobulinaemia (polyclonal, characteristic of chronic inflammatory disorders or autoimmunity, and monoclonal, often with an associated malignant disorder) and hypogammaglobulinaemia (including immunodeficiency, immune paresis secondary to malignancy and protein loss). Where dysgammaglobulinaemia is noted in the elderly the most useful laboratory tools to help discern the pathogenesis are serum and urine electrophoresis, autoantibody investigations and measurement of liver and renal function.

Multiple sclerosis and occult gluten sensitivity.

Two atypical patients with a multiple sclerosis (MS)-like illness and evidence of occult celiac disease (CD) were managed by the authors. This prompted screening of a further 49 unselected MS cases for serologic evidence of CD. IgA anti-endomysial antibody was found in one case (2%). IgG anti-gliadin antibody was found in 12% of patients and 13% of blood donors. Anti-gliadin antibody (especially IgG isotype) can be a nonspecific finding.