Anaphylaxis Unpredictable Can Occur In Anyone Any Time Biology Essay

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Anaphylaxis is unpredictable and can occur in anyone at any time. The rate of occurrence of anaphylaxis is increasing and it is vitally important to understand the potential triggers, mechanisms, and patient specific risk factors to determine the potential risk of an acute anaphylactic attack (Lez-pe´ rez et al, 2010)

Anaphylaxis is a type 1 hypersensitivity reaction mediated by IgE (Fig 1.0) but can also be anaphylactoid (not involving IgE and generally triggered by drugs) or physical (triggered by physical factors such as exercise, heat or cold). Anaphylactic reactions are rapid, and usually occur within minutes, or at most an hour after exposure to the offending allergen (Estelle & Simons, 2010).

Clinical diagnosis of anaphylaxis involves pattern recognition and a meticulous history of an exposure or event preceding characteristic anaphylactic symptoms (despite laboratory investigations indicating that test results are within normal limits). The number of potential anaphylaxis triggers can be extensive, common triggers include foods, medications, and insect stings. Raised total serum IgE levels and positive skin prick tests are useful to confirm prior sensitisation but are not always specific to the causative trigger of anaphylaxis. Patient-related risk factors for anaphylaxis include age, pre-existing diseases (e.g. asthma), medications (e.g. ß-blockers and angiotensin-converting enzyme (ACE) inhibitors), infections, stress and defects in mediator degradation pathways (Estelle & Simons, 2009).

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The only current treatment to prevent anaphylaxis is dependant on management of patient related risk factors as well as strict avoidance of confirmed relevant allergen or triggers. In some cases, immunomodulation may be used to desensitise patients, e.g., subcutaneous venom immunotherapy to prevent Hymenoptera sting-triggered anaphylaxis, which in some cases can provide a potentially curative treatment. During an acute anaphylactic episode, epinephrine self-injectors (EpiPen) are the first choice of treatment to treat an anaphylactic episode (Estelle & Simons, 2009).

Idiopathic Anaphylaxis

Despite rigorous investigations some patients with anaphylaxis can present with symptoms for which no allergic or physical trigger can be identified. Having excluded rare conditions such as mastocytosis or endocrine disorders the condition is termed idiopathic anaphylaxis (Estelle & Simons, 2010).

Omega 5 gliadin

Individuals suffering from wheat-dependant, exercise-induced anaphylaxis (WDEIA) develop IgE directed against wheat Omega 5 gliadins and wheat low-molecular weight glutenin subunits (LMW-GS). WDEIA is characterised by the development of an anaphylactic episode triggered when wheat is ingested prior to physical activity. The mechanism for this reaction is unknown but currently specific IgE to Omega 5 gliadin has proven to be a useful tool in aiding the diagnosis of these patients. However, certain studies have found that specific IgE to Omega 5 gliadin via solid phase reaction (such as Phadia Immunocap 250) cannot distinguish between wheat sensitisation and genuine clinical reactivity to the allergen (Beyer et al, 2008; Ito et al, 2003).

Investigations into the causes of these acute conditions can be time consuming, invasive, stressful for all parties involved and expensive. Therefore, in order to solve this diagnostic problem, a cost effective method of identifying previously unrecognised triggers needs to be found which is rapid, sensitive and specific which offers a wide panel of tests to aid confirmation of an anaphylactic trigger .

Current methods of allergen detection

Currently meticulous patient history, skin prick testing and serological test systems using allergen extracts (prepared from various crude whole allergen preparations such as pollens, foods, animal dander, and insect venoms) are used to narrow down the specific allergen (Deinhofer et al, 2004; Harwanegg and 2004). However, because whole extract is used, it is difficult to determine if the patient is sensitised to the whole allergen or a component of that given allergen. The significance being that individuals who are positive for a specific allergen may either have genuine sensitisation to that allergen or have developed an immunological cross-reactivity to structurally related allergen molecules present in a variety of other allergens, e.g., the cross-reactive component profilin (Sicherer and Leung et al, 2010; Barber et al, 2009). The gold standard is challenge testing, which is sometimes performed but is associated with a significant clinical risk. Therefore, a method of breaking down these allergen sources into their component allergens may prove more useful in extending our knowledge of which allergens are best avoided by patients (Jahn-Schmid et al, 2003).

Component resolved diagnostics (CRD) by Microarray biochip technology (Phadia ISAC).

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ImmunoCAP ISAC is a multiplex semi-quantitative in vitro diagnostic tool and is based exclusively on allergen components. This multiplex assay allows for measurement of specific IgE antibodies to many allergen components using only 20μl of serum or plasma (Fig 2.0). Up to 103 purified native and recombinant allergens are immobilized on a solid support biochip (Phadia ImmunoCAP ISAC (2008).

Previous work in this field

To our knowledge only one previous study has looked at the role of an allergy microarray assay in idiopathic anaphylaxis. A group from the University Hospital of Wales (Cardiff) has recently studied sixty idiopathic anaphylaxis patients from a number of hospitals in the UK. The patients serum was hybridised to an ISAC microarray1-5 (Phadia) containing 103 purified native and recombinant allergens. Out of the sixty patients, thirty ISAC results were considered to have provided additional useful clinical information. In twenty two of the patients the ISAC array revealed previously undetected allergen sensitisation. In fourteen of the patients it was considered a high likelihood that the additional sensitisations were related to the patient's symptoms and could potentially change the clinical management of the patient (Heaps et al, 2010).

Methodology

Patients

A cohort of patients diagnosed with idiopathic anaphylaxis, severe urticaria/angioedema or WDEIA in the peninsula allergy clinic, Derriford Hospital will be included in this study. It is planned to study anything from Ten to thirty patients in each group but the exact number will depend on funding. The diagnosis of idiopathic anaphylaxis will have been made after extensive history taking and negative investigations including specific IgE testing and/or skin prick testing.

Blood Samples

Blood samples will be collected from patients diagnosed with idiopathic anaphylaxis, severe idiopathic urticaria/ angioedema and WDEIA as part of their routine clinical work-up. Samples will be collected in SST gold top vaccutainer tubes containing clot activator and gel for serum separation. Samples will be stored at -20°C until required.

Fig 2.0 Microarray technique

ImmunoCAP ISAC slides are placed in to a humidity chamber

20μl of each sample/control serum

is pipetted onto one reaction site.

Incubated at room temperature for 120 minutes

Wash for 10 minutes with wash solution + further 5 minutes with purified water using a washing dish and magnetic stirrer

Air dry then add 20μl of Detection antibody solution

Wash for 10 minutes with wash solution + further 5 minutes with purified water using a washing dish and magnetic stirrer

Air dry then read

(Fig 2.0) Adapted from Phadia (2008) ImmunoCAP ISAC IgE assay kit insert.

Fig 3.0 Basic schematic of ImmunoCAP ISAC slide.

Patient Serum

Detection antibody solution

BioChip

The procedure is followed by image acquisition using an appropriate microarray scanner such as a confocal laser scanning device CapitalBio LuxScanâ„¢ 10K microarray scanner provided by University Hospital of Wales (Cardiff). The ISAC Standardized Units (ISU) are determined and the test results are analyzed with proprietary software (MIA - Microarray Image Analysis Software) (Fig 3.0)

Fig 3.0 Example of scanner reading of ImmunoCAP ISAC chips.

Graphic taken from: ImmunoCAP ISAC in vitro test procedure kit insert.

Patient follow-up

All the patients included in the study will be followed up in the allergy clinic as per normal practice. Any patients with a new positive ISAC micro array result or Omega-5-gliadin positive will have a full history re-taken in light of the new information. Where appropriate, additional skin prick testing, specific IgE testing and challenge testing will be performed to determine the clinical significance of the findings.

Analysis

Table 1.0 provisional time line to complete project.

Activity

Estimated time period

Obtain ethics and patient consent

8 weeks

Collect patient samples and store at -20°C.

8 weeks

Collect patient history and attend allergy/SPT clinics

4 weeks

Analyse information

4 weeks

Perform Phadia ISAC and read results using specialist software and scanner.

1 week

Analyse Data

4 weeks

Patients with new positive allergen results and Omega 5 gliadin positive will be recalled to clinic

12 weeks - repeat history and possible SPT.

Analyse Data

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8 weeks

Write up results

12 weeks

Data Analysis

Patient data (diagnosis, specific IgE results, skin prick test results and challenge results) performed by the chief investigator Dr E Kaminski and will be anonymised and stored in a secure data base.

Microarray Image Analysis Software (MIA) will be used to interpret the results from the Immunocap ISAC chips provided by University Hospital of Wales (Cardiff) and the data from the Immunocap 250 will be interpreted by a HPC registered biomedical scientist. This will be stored in a secure database.

Summary

This study hopes to collect and analyse serum from previously diagnosed patients with idiopathic anaphylaxis and severe urticaria or angiodema from Derriford hospital and the University Hospital of Wales, Cardiff, UK and analyse their serum using Phadia ISAC. These results will then be analysed and compared to previous specific IgE and skin prick test results. Any new allergens identified from the Phadia ISAC in the patient cohort, will be analysed and the patients will be brought back to clinic and the clinical relevance of these findings will be determined from further history taking, specific IgE testing and skin prick testing if appropriate. If necessary, patients will be challenged with the newly identified allergens. If further funding is approved this study may look at a cohort of patients with brittle asthma. The discovery of any new allergic triggers discovered could radically transform the lives of these patients and potentially even save their lives.