BasophilBasophil ActivationActivation TestingTesting An in vitro test for the diagnosis of IgE and non-IgE mediated allergies Alpha Laboratories Ltd. Diagnostics 2018

A Safer Allergy Testing Alternative for Your Patients

A Safer Alternative for Allergy Testing 2 A Proposed Patient Pathway for Allergy 2-3 Introducing Flow CAST® Basophil Activation Test 3

Allergy Testing- Without the Risk 4 Case Study - University Hospital Southampton

Diagnosing Drug Allergy: A Rational Approach 5-6 Expert Opinion - Dr Cathy van Rooyen

BÜHLMANN Allergens List 7-8 A Safer Alternative for Allergy Testing

The physical symptoms of allergy can be similar BAT is a safer alternative to a provocation to those of a sensitivity or intolerance. Roughly challenge for your patient. Offering BAT before 20% of the population experience symptoms a challenge test will significantly reduce the that make them believe they have a food allergy. number of patients having to undertake a However, it is estimated that actually only 1-10% provocation challenge. For example a peanut BASOPHIL ACTIVATION TESTING of adults and children do have a food allergy1. study undertaken by Santos et al. has shown a 67% reduction in the need for oral food 3 INTRODUCTION Less than 10% of people who think they are challenge by using BAT as the primary test. allergic to penicillin are truly allergic. If someone Eliminating the need for a provocation challenge For over 40 years Alpha Laboratories is labelled as penicillin allergic they are likely to improves patient safety and comfort. be treated with broad-spectrum antibiotics2. An has been providing quality supply incorrect label of penicillin allergy may lead to The true value of BAT lies in its high specificity, and support to UK and international inappropriate use of broad-spectrum antibiotics, (reaching 100% in some studies3). A positive laboratory and clinical scientists. This increasing the threat of antibiotic resistance2. BAT result confirms allergy with a high degree family run company is led by its vision of certainty, resulting in fewer of the more to “find new ways to help science It is vital to distinguish which patients are dangerous positive provocation challenges being improve people’s lives through its truly allergic from those who are not. Basophil performed. evolving range of specialist Diagnostic Activation Testing (BAT) can be a helpful and Laboratory Products.” diagnostic tool to achieve this. References 1. Allergy UK. Allergy Prevalence: Useful facts and Studies have shown that BAT has a higher figures. Through worldwide connections sensitivity and specificity than standard skin prick 2. NICE Clinical Guideline 183. Drug Allergy: Diagnosis and relationships with leading tests (SPT) and specific IgE tests (sIgE), making and management of drug allergy in adults, children and young people. September 2014. manufacturers, established and it a useful second line test if SPT and sIgE results emerging suppliers, Alpha Laboratories 3. Santos AF, Shreffler WG. Road map for the clinical are unable to offer a confirmed result. application of the basophil activation test in food sources the best products to help allergy. Clin Exp Allergy. 2017; 47:1115–1124. https:// solve the problems you face in your doi.org/10.1111/cea.12964 laboratory or clinic every day.

Bringing you solutions that: ■ Improve the quality of your results A Proposed Patient Pathway for Allergy ■ Save time ■ Lower overall costs ■ Maximise your uptime & efficiency Clinical History ■ Improve your supply chain ■ Reduce health and safety risks ■ Support better patient outcomes in Skin Prick Test (SPT) clinical environments and/or Specific IgE Test (sIgE) Our partnership with BÜHLMANN Laboratories AG extends over 10 years and we are proud to be their exclusive Negative UK distributor. BÜHLMANN is an Allergy confirmed Positive Results if clinical history Allergy excluded is convincing innovative specialist in vitro diagnostics manufacturer with a selection of unique, high-quality assays for routine Equivocal clinical use.

Allergy is one area of focus for Basophil Activation BÜHLMANN and Alpha Laboartories. Test (BAT) Where specific IgE fails to demonstrate absolute diagnostic reliability, especially in more complex reactions such as drug, venom or food allergies we have Allergy confirmed Positive Results Negative the solution - The complete Cellular Allergy diagnostics package. Provocation Challenge

Cover image background photo by Joel Filipe on Unsplash.com Allergy confirmed Positive Results Negative Allergy excluded

2 Introducing Flow CAST® Basophil Activation Testing Made Easy

The Flow CAST® system, manufactured by Upon response to a purified allergen which Flow CAST® Data Acquisition BÜHLMANN Laboratories, represents a is added to the sample, sensitised basophils significant step forward in in vitro allergy degranulate and express the cell surface marker testing. Using a unique dual marker CD63. This is detected by a FITC-fluorescence combination (CCR3 and CD63) it allows the labelled monoclonal antibody. CD63 is currently simple detection of Basophil Activation on the best-validated basophil activation marker whole blood samples via flow cytometry. Flow directly linked to basophil degranulation. CAST BAT can be used to identify immediate The Flow CAST kit can identify both IgE and type allergic reactions and hypersensitivities to non-IgE mediated allergies, making it ideal suspected allergens. for determining allergies to anaesthetics, antibiotics and NSAIDs. Basophils constitutively express the eotaxin receptor CCR3 allowing for a robust and accurate selection of cells with flow cytometry. The Flow CAST kit is quick and easy to use, CCR3 is detected with a PE-fluorescence with results obtained in ~60 minutes. Samples labelled monoclonal antibody. This negates the can be analysed up to 48 hours after collection Figure 1: Three discrete populations; requirement for lengthy and labour intensive (up to 24 hours for drug responses). The lymphocytes, monocytes and leukocyte isolation steps. kit contains all of the reagents and controls granulocytes in FSC/SSC histogram. needed to run the test.

An extensive range of over 150 standardised allergens spanning food, drug, insect venoms, inhalants and food additive allergies are available to purchase separately.

Find out more about Flow CAST kits (Product Code FK-CCR) and purchase online at ww.alphalabs.co.uk/flowcast

Figure 2: Selection of basophilic cells CCR3pos / SSClow Clinical History

Skin Prick Test (SPT) and/or Specific IgE Test (sIgE)

Negative Allergy confirmed Positive Results if clinical history Allergy excluded is convincing

Equivocal Figure 3: Patient Background (PB) with Stimulation Buffer only

Basophil Activation Test (BAT)

Allergy confirmed Positive Results Negative

Provocation Challenge

Allergy confirmed Positive Results Negative Allergy excluded Figure 4: Positive Control (PC) with Stimulation control anti-FceRI Ab Basophil Activation Testing Allergy Testing- Without the Risk Yoon Tak Chin, Bryan Fernandes, Adnan Mani, Clive McLean, Efrem Eren. University Hospital Southampton NHS Foundation Trust, Laboratory medicine, Division of Diagnostics and Therapy Department of Immunology.

In some cases of allergens that cause Ordinarily, drug allergy testing to ibuprofen immediate complex reactions, such as would involve a drug provocation those to drug allergens, specific IgE fails challenge while drug allergy testing to penicillin to give absolute diagnostic reliability. would involve a series of skin prick Whilst drug provocation tests remain the and intradermal tests. gold standard for diagnosis of immediate drug allergies, they are very time intensive However, in view of the severity of the reaction, procedures associated with the significant the young girl, her mother and our Paediatric risk of severe and potentially fatal colleagues were understandably reluctant to reactions. Likewise, skin prick and intra- perform these tests. dermal testing are slow and risk severe  reactions. In vitro tests such as specific Instead a blood sample was taken and tested IgE to drugs offer a limited range of drugs using the BÜHLMANN Flow CAST® BAT kits. that can be tested. Results for ibuprofen and amoxicillin were both Basophil Activation Tests (BAT) provide an positive. BAT was negative for other agents References 1. Ebo DG, Bridts CH, Hagendorens MM, Mertens CH, alternative test that does not carry risks to including aspirin, penicillin major determinants De Clerck LS, Stevens WJ. Flow-assisted diagnostic the patient. They have emerging potential and penicillin minor determinants. management of anaphylaxis from rocuronium in assisting in the clinical work-up and bromide. Allergy. 2006;61:935-9. diagnosis of immediate drug allergies. This data helped define the Paediatric Team’s 2. Gamboa PM, Sanz ML, Caballero MR, Antépara I, management plan, without putting the young Urrutia I, Jáuregui I, González G, Diéguez I, De Weck Here a team from the Department girl through a long procedure or the risk of a AL. Use of CD63 expression as a marker of in vitro of Immunology, University Hospital severe reaction. basophilactivation and leukotriene determination Southampton NHS Foundation Trust, in metamizol allergic patients. Allergy. 2003;58:312-7. 3. Gómez E, Blanca-López N, Torres MJ, Requena describe an example of their experience There are studies showing that BAT can be G, Rondon C, Canto G, Blanca M, Mayorga C. using BAT in a clinical setting. helpful in diagnosing immediate allergy to some Immunoglobulin E-mediated immediate allergic Our clinical team had a case of a 12-year old drugs, particularly muscle relaxants such as reactions to dipyrone: value of basophil activation test girl who developed anaphylaxis immediately rocuronium (sensitivity 91.7% and specificity in the identification of patients. Clin Exp Allergy. after taking ibuprofen and amoxicillin. In 100%)1 and NSAIDs (sensitivity 42.3%-56.7% 2009;39:1217-2 this situation we found use of the Basophil and specificity 83%-100%)2&3. Further studies Activation Test (BAT) has been helpful. are needed to evaluate the usefulness of BAT for other drugs. 

4 Diagnosing Drug Allergy: A Rational Approach by Dr Cathy van Rooyen, MBChB, MMed Path, FRCPath (Virol) Allergy, Immunology & Virology Pathologist at AMPATH Pathology, South Africa

Ideally BAT should be used in combination Introduction with other tests for optimal sensitivity. Negative Many patients experience adverse reactions results should always be followed up with to drugs, but most of these are predictable, additional testing. BAT is best described for dose dependent and don’t involve the β-lactam antibiotics, NSAIDS, myorelaxants, immune system. They include side-effects, contrast media and local anaesthetics. drug interactions and toxic effects due to 1 overdosing. Some patients may also be very Approach to Drug Allergy Diagnosis: sensitive to the normal pharmacological As drug allergy diagnosis may be quite effect of a drug or have a genetic or enzyme complicated, it is essential to follow a rational deficiency affecting the metabolism of certain approach utilising available resources. The drugs. In vivo1,5 skin tests6 or drug provocation tests7,8 flow chart on page 6 shows an example of an can be performed, but these can be time approach followed in private practice in South Drug allergy is an immunologically mediated consuming, costly and potentially harmful. Africa. This could be adapted to suit local reaction that is specific to a particular drug and Drug provocation tests should be performed resources and test menus. 2 reoccurs on subsequent exposure to that drug. in specialist allergy centres under the highest Many different immunological mechanisms safety conditions. A risk-benefit analysis should Concluding Remarks may be involved, including IgE, basophil, be carried out first for a patient with a history It is essential to correlate all test results with eosinophil, cytotoxic/complement, immune of anaphylaxis to the tested drug, severe your patients’ clinical history. No single drug complexes and T-cell mediated reactions.1,3 concurrent illness or pregnancy. allergy test is sensitive enough to rule out drug allergy in a patient with a convincing history These immunologic drug reactions may 1,5 A number of in vitro laboratory tests are also of drug allergy. A combination of drug allergy necessitate changes in therapy or may be life- available and are preferable, as there is no risk tests provides the highest sensitivity. Once a threatening, therefore a definitive diagnosis to the patient. diagnosis of drug allergy has been confirmed, it and the identification of safe alternatives are is important to identify a safe alternative to the usually required. Drug-specific IgE analysis is one option but drug in question. If no alternative can be found offers a limited availability of allergens and lacks and the drug is essential, drug desensitisation sensitivity. Although it may have good specificity should be considered. (90%), negative tests should always be followed up with additional testing in patients with a You can read Dr van Rooyen’s full report at: convincing history of drug allergy. www.alphalabs.co.uk/allergydiagnosis

T-cell proliferation Lymphocyte transformation References: tests (LTT) should only be used in expert 1. Demoly P, Adkinson NF, laboratories and are often still only a research Brockow K, Castells M, Chiriac AM, et al. tool. They need to be used in combination with International Consensus on drug allergy. Allergy 2014;68:420-437. other tests, as they may lack sensitivity. 2. Baldo BA, Pham N. Drug Allergy: Clinical aspects, Diagnosis, Mechanisms, Structure-activity A generally accepted classification of The mast cell tryptase test is used to confirm relationships. Springer: New York Heidelberg. 2013. immunologic drug reactions (drug allergy) anaphylaxis and indicates the involvement of 3. Pichler WJ. Delayed drug hypersensitivity reactions. is based upon the timing of the appearance mast cells, whatever the cause of degranulation. Ann Intern Med 2003; 139:683-693 of symptoms. Reactions may be immediate1, 4. Blanca M, Romano A, Torres MJ, Fernandez J, occurring within 1-6 hours after drug Mayorga C, et al. Update on the evaluation of administration. These are often IgE-mediated hypersensitivity reactions to betalactams. Allergy and sometimes basophil-mediated or other 2009;64:183-193. non-IgE dependant reactions. Delayed type 5. Pichler WJ. An approach to the patient with drug allergy. Up To Date. April 2015. Available from: reactions3 typically occur after 24 hours and https://www.uptodate.com/contents/an-approach- often appear following multiple doses of to-the-patient-with-drug-allergy. treatment, typically after days or weeks of 6. Brockow K, Garvey LH, Aberer W, Atanaskovic- administration. The timing of the reaction Morkovic M, Barbaud A, et al. Skin test can help guide the clinician in the choice of concentrations for systemically administered drugs diagnostic technique.4 – an ENDA/EA-ACI Drug allergy Interest Group position paper. Allergy 2013;68:702-712. How Do I Diagnose a Drug Allergy? 7. Joint task force on practice parameters; American The Basophil Activation Test (BAT) can be Academy of Allergy, Asthma and Immunology; Joint The aim is to establish or disprove a causal used as a first-line test for drug allergy. It is a Council of Allergy, Asthma and Immunology. Drug relationship between the drug and the patient’s popular option for use in allergy centres and allergy: an updated practice parameter. Ann Allergy reaction and there are a number of tools can be included as part of a routine diagnostic Asthma Immunol 2010;105:259-273. available to investigate this. algorithm. The specificity of BAT for drug allergy 8. Mirakian R, Ewan PW, Durham SR, Youlten LJ, Dugue is generally high (93%). Sensitivity depends on P, et al. BSACI guidelines for the management of the drug being tested but usually exceeds 50%. drug allergy. Clin Exp Allergy 2009;39:43-61.

Find out more at www.alphalabs.co.uk/allergy 5 Basophil Activation Testing A Diagnostic Algorithm for Drug Allergy Diagnosis

History compatible with drug allergy

Immediate Timing of reaction Delayed

Skin tests available? Skin tests available? -Skin prick tests -Intradermal tests (delayed reading) -Intradermal tests -Patch tests

Yes No No Yes

Results Neg Drug specific lgE - LTT Neg Results -BAT - BAT

POS Results Results POS

Drug allergy Neg Drug allergy confirmed confirmed

Drug important? Drug provocation available?*

No Yes

- Use alternate drug Results - Re-administration under surveillance -Drug desensitisation Neg POS

Drug allergy Drug allergy excluded confirmed

*Drug provocation contra-indicated in patients with a history of severe reactions e.g. SJS, TEN, DRESS or vasculitis.

6 BÜHLMANN Allergens Quality Controlled Allergens and Mixes Optimised for in vitro use in the Cellular Allergy Assays - www.alphalabs.co.uk/allergens

DRUGS BAG2-C55 ACETAMINOPHEN INSECT VENOMS BAG2-C56 MEFENAMIC ACID BAG2-C57 PHENYLBUTAZONE BAG2-C58 PROPYPHENAZONE BAG2-C59 DIPYRONE / METAMIZOLE BAG2-CNAP NAPROXEN BAG2-CTRA TRAMADOL BAG2-C114 SODIUM SALICYLATE Myorelaxants BAG2-CATR ATRACURIUM BAG2-CCAT CISATRACURIUM BAG2-CMIV MIVACURIUM BAG2-CPAN PANCURONIUM

Antibiotics BAG2-CPRO PROPOFOL BAG2-I1 HONEY BEE VENOM BAG2-C1 PENICILLIN G BAG2-CROC ROCURONIUM BAG2-I1CHK HONEY BEE VENOM CHECK BAG2-C11 BENZYLPENICILLOYL-POLY- BAG2-CSUX SUXAMETHONIUM LYSIN, PPL BAG2-CVEC VECURONIUM BAG2-I3 YELLOW JACKET VENOM BAG2-C12 BENZYLPENICILLIN +-ACID, BAG2-CNBX NEUROMUSCULAR BAG2-I3CHK YELLOW JACKET VENOM MDM BLOCKERS MIX (CATR, CCAT, CHECK BAG2-C2 PENICILLIN V CMIV, CPAN, CPRO, CROC, BAG2-I75 EUROPEAN HORNET VENOM CSUX, CVEC) BAG2-C3 CEPHALOSPORIN C BAG2-I77 EUROPEAN PAPER WASP Local Anesthetics VENOM BAG2-C31 CEFAMANDOLE BAG2-CART ARTICAINE BAG2-C32 CEPHAZOLIN BAG2-CBUP BUPIVACAINE BAG2-C33 CEFUROXIME INHALANTS BAG2-CMEP MEPIVACAINE BAG2-C34 CEFACLOR Grasses BAG2-CLID LIDOCAINE BAG2-C35 CEFTRIAXONE BAG-G2 BERMUDA GRASS Contrast Media BAG2-C36 LEVOFLOXACIN BAG-G3 ORCHARD GRASS BAG2-CIOBI IOBITRIDOL BAG2-C61 SULFAMETHOXAZOLE BAG-G5 PERENNIAL RYE GRASS BAG2-CIODI IODIXANOL BAG2-C62 TRIMETHOPRIM BAG-G6 TIMOTHY GRASS BAG2-CIOHE IOHEXOL BAG2-C75 TETRACYCLINE BAG-G12 CULTIVATED RYE GRASS BAG2-CIOME IOMEPROL BAG2-C76 DOXYCYCLINE BAG-GX1 6-GRASS MIX (G3, G5, G6, BAG2-CIOPA IOPAMIDOL BAG2-C81 CIPROFLOXACIN MEADOW FESCUE, MEAD- BAG2-CIOPR IOPROMIDE OW GRASS, VELVET GRASS) BAG2-C82 MOXIFLOXACINE BAG2-CIOXA IOXAGLATE Weeds BAG2-C203 AMPICILLIN Antiseptics BAG-W1 COMMON RAGWEED BAG2-C204 AMOXYCILLIN BAG2-CCHX CHLORHEXIDINE BAG-W6 MUGWORT BAG2-CCLA CLARITHROMYCINE Proton Pump Inhibitors (PPI) BAG-W9 RIBWORT/PLANTAIN BAG2-CCLAX CLAVULANIC ACID AMOX (CLAVULANIC ACID BAG2-COME OMEPRAZOLE BAG-W19 PELLITORY AMOXYCILLIN 20/80) BAG2-CESO ESOMEPRAZOLE BAG-WX1 RAGWEED MIX BAG2-CCLIN CLINDAMYCINE BAG2-CPANT PANTOPRAZOLE (W1, GIANT RAGWEED) BAG2-CERY ERYTHROMYCINE BAG2-CLAN LANSOPRAZOLE Trees BAG2-CRIF RIFAMPICINE Beta Blockers BAG-T3 COMMON BIRCH Analgesics BAG2-CBIS BISOPROLOL BAG-T4 HAZEL BAG2-C51 LYS-ASPIRIN BAG2-CMET METOPROLOL BAG-T7 OAK BAG2-C52 DICLOFENAC ACE inhibitors BAG-T9 OLIVE BAG2-C53 IBUPROFEN BAG2-CRAM RAMIPRIL ...... continued BAG2-C54 INDOMETHACIN

Find out more at www.alphalabs.co.uk/allergy 7 Basophil Activation Testing BÜHLMANN Allergens continued

Moulds Fish & Meat Cereals BAG-M1 PENICILLIUM BAG-F3 CODFISH BAG-F4 WHEAT BAG-M2 CLADOSPORIUM BAG-F23 CRAB (CALLINECTES SAPI- BAG-F79 GLUTEN - Wheat BAG-M3 ASPERGILLUS DUS) BAG-F98 GLIADIN - Wheat BAG-M5 CANDIDA ALBICANS BAG-F24 SHRIMP BAG-F5 RYE FLOUR BAG-M6 ALTERNARIA BAG-P4 ANISAKIS BAG-F6 BARLEY FLOUR BAG-F26 PORK BAG-F7 OAT FLOUR BAG-F27 BEEF BAG-F8 MAIZE BAG2-GAL ALPHA-GAL-HSA BAG-F9 RICE Fruits & Vegetables BAG-F45 BAKER`s YEAST BAG-F25 TOMATO Spices BAG-F31 CARROT BAG2-F218 PAPRIKA BAG-F33 ORANGE BAG2-F281 CURRY BAG-F85 CELERY BAG2-F279 CHILLI BAG-F95 PEACH BAG2-F317 CORIANDER BAG2-F47 GARLIC BAG2-FCUR CURCUMA BAG2-PRUP3 nPRU P 3 LTP PEACH Food Mixes BAG2- rMAL D 1, APPLE* BAG-FX1 FOOD MIX (F1, F75, F2, F3, MALD1 F4, F13, F14, F17, F24) Seeds, Beans & Nuts Mites, Animals & Insects BAG-F10 SESAME FOOD ADDITIVES BAG-D1 HOUSE DUST MITE BAG-F13 PEANUT BAG2-C103 TARTRAZINE BAG-D2 AMERICAN HOUSE DUST BAG2-C105 METHYLENE BLUE MITE BAG2- nARA H 1 PEANUT ARAH1 BAG2-C111 SODIUM-BENZOATE BAG-D70 ACARUS SIRO BAG2- nARA H 2 PEANUT BAG2-C112 SODIUM-NITRITE BAG-DX1 STORAGE MITE MIX ARAH2 BAG2-C113 POTASSIUM-METABISULFITE BAG-E1 CAT EPITHELIUM BAG2- nARA H 6 PEANUT BAG2-C114 SODIUM-SALICYLATE BAG2-FELD1 rFEL D 1, CAT* ARAH6 BAG2-CE104 QUINOLINE YELLOW BAG-E2 DOG EPITHELIUM BAG-F14 SOYBEAN BAG2-CE110 SUNSET YELLOW FCF Inhalant Mix BAG-F17 HAZELNUT BAG2-CE122 CHROMOTROPE FB BAG-IX1 INHALANT MIX (GX1, G12, BAG-F20 ALMOND T3, T4, W6, W9, M6, D1, BAG2-CE123 AMARANTH BAG-F202 CASHEW NUT D2, E1, E2) BAG2-CE124 NEW COCCINE BAG-F203 PISTACHIO NUT BAG2-CE127 ERYTHROSINE BAG-F256 WALNUT BAG2-CE131 PATENT BLUE V OCCUPATIONAL BAG2-F36 COCONUT BAG2-CE132 INDIGO CARMINE BAG-K82 LATEX BAG2-CE151 BRILLIANT BLACK BN BAG2-CE202 SORBIC ACID FOOD BAG2-CE466 CARBOXYMETHYLCELLU- LOSE Egg BAG2-CE621 GLUTAMATE BAG-F1 EGG WHITE BAG2-C101 FOOD COLORANTS I MIX BAG-F75 EGG YOLK (CE104, CE110, CE122, Milk CE123, CE124) BAG-F2 COW'S MILK BAG2-C102 FOOD COLORANTS II MIX BAG-F76 ALPHA-LACTALBUMIN (CE127, CE131, CE132, CE151) BAG-F77 BETA-LACTOGLOBULIN Find out more and order online at BAG-F78 CASEIN www.alphalabs.co.uk/allergens

40 Parham Drive, Eastleigh, Hampshire, SO50 4NU, UK Tel: 023 8048 3000 | Email: [email protected] Web: www.alphalabs.co.uk/allergy Registered in England 1215816