22 nd Nov 2017 Dr Pravin Hissaria Basophil Activation Test (BAT)ting for better allergy diagnosis Clinical Immunologist and Immunopathologist (Staff Specialist) SA Pathology/ Royal Adelaide Hospital
Allergy - Type 1 Hypersensitivity reactions RCPA Immunology QAP Sensitisation Clinical Allergy
Laboratory testing for Allergy diagnosis BAT Total IgE RCPA Immunology QAP Sensitisation Clinical Allergy
History of diagnostic allergy testing
Current Status of Allergy testing sige testing confirms allergy in individuals with a good history of reactions (does not diagnose or predict) Serum tryptase confirms a severe allergic reaction but not the cause Current Gold standard Challenge with the allergen
Current challenges in food allergy diagnosis Food allergy Diagnosis when multiple foods ingested Hospital waiting times for conducting challenges Risks involved for subjects Drug Allergy Diagnosis when multiple drugs involved Delabelling Issues with challenges No lab testing available for most drugs Insect sting allergy Similar issues Outcome of desensitisation therapy
Principle of basophil activation test
Technical aspects Sampling and preservation of Basophils Should be processed within 3-4 hours after blood collection Similar profile seen with EDTA or heparin Can be stored at room temp but preferrably at 4 C till processing
Gating strategies for basophils Initially surface IgE (FlowCAST assay) CCR3 based gating (with or without CD3) Flow2CAST assay CRTH2
Basophil Gating CD123 + / HLA DR-ve Clinical and Experimental Allergy 2008
Other technical considerations Priming with IL-3 before stimulation Positive control Anti IgE, fmlp Negative control Non-responders 5-10% Concentration of Allergen preferably at least 3 log concentrations Inter and Intra operator variability
Interpretation of BAT results Cytometry B 2014
CD63% activation Clinical Applications of BAT : Food allergy Cashew BAT results (March 2017) 20yo male Oral Sx to cashew age 10 SPT age 17 positive for cashew and pistachio No asthma, carries Epipen but wants to join ADF 100 90 80 70 60 50 40 30 20 10 0 86.5 1.3 2.5 37.7 63.2 17.2 2017 SPT cashew 6mm, pistachio 5mm sige cashew <0.35, pistachio 0.44 Cashew BAT positive OFC June 2017 Incremental doses starting from 3mg, half-log increases to 6g (4 cashews) (total dose 10g) Challenge ceased due to development of abdominal pain and acute rhino-conjunctivitis Assessment and recommendation: cashew allergy, but relatively mild and does not need to carry an Epipen
CD63 activation% CD63 activation% Initial BAT results (August 2015) 23yo male Asthma, eczema, AR 60 50 51.4 2011 Mild anaphylactic reactions suspected to peanut in 2009 and 2011 SPT: peanut 14mm, cashew 2mm 40 30 20 10 0 29.5 1.1 31.9 17.7 2013 Throat Sx with peanut oil SPT: peanut 7/20 cashew 2.5/3 2015 1x mild-mod reaction?nut ingestion SPT: peanut 21/40, cashew 15/20, pistachio 6/15 sige peanut 2, rarah2 1.8, rarah 1/3/8/9 <0.35, cashew <0.35 OFC: tolerated ½ peanut. Desensitisation: tolerated 12 peanuts BD (minor abdo pain during updosing phase) Repeat BAT results (June 2017) 90 80 70 60 50 40 30 20 10 0 83.3 4.2 4.2 2.8 7 43.9 3.8 7.5 82.4 79.1 2017 Interrupted desensitisation sige peanut 3.1, cashew 1.1
Drug Allergy Patient Sex Age Culprit Drug Time interval between drug intake and allergic reaction Reaction Type Time interval between allergic reactions and BAT BAT results towards the culprit Drug Type 1 Hypersensitivity towards the culprit drug as confirmed by strong History ± DPT ± ST 1) ET F 45 Cephalexin 10 min Anaphylaxis 10 months Negative Negative (ST) 2) MM M 55 Cefazolin Immediate Anaphylaxis 5.5 Months Positive (++) Positive (SH and ST). 3) DW F 60 Cefaclor 2.5 hours Anaphylaxis 7.5 Months Negative Negative (ST) 4) IZ F 68 Ceftriaxone A few hours Pruritic Hives 5 Months Negative Negative (ST and DPT) 5) AH F 64 Cephalexin 20 min Anaphylaxis 10 Months Negative Positive (ST) 6) AM F 73 Ceftriaxone Immediate Anaphylaxis 4 days Positive (++) Positive (SH) 7) TS F 51 Cephalexin Immediate Anaphylaxis NM Positive (++) Positive (ST) 8) EE F 21 Cefazolin Immediate Anaphylaxis 7 Months Negative Negative (ST and DPT) 9) AH M 46 Cephalexin 5-15 mins Anaphylaxis 7 months Negative Negative (ST and DPT) 10) JB F 57 Cephalexin Immediate Anaphylaxis >40 years Negative Negative (SH and ST) 11) RS M 75 Ceftriaxone Few hours urticaria 7 months Negative Negative (ST) 12) MM (Repeat) M 55 Cefazolin Immediate Anaphylaxis 13.5 Months Positive (++) Positive (SH and ST) 13) VS M 60 Cephalexin Immediate Anaphylaxis 11 Months NON-RESPONDER Positive (ST &DPT) 14) RR F 71 Ceftriaxone Immediate pruritis 3months Negative Negative (ST &DPT) 15) RR (Repeat) F 71 Ceftriaxone Immediate pruritis 3months Negative Negative (ST &DPT) 16) JZ F 36 Ceftriaxone Immediate Anaphylaxis 8 months Negative Negative(ST) 17) VH F 56 Cephalexin 20 min Anaphylaxis 8 months Negative Negative(ST) 18) FC M 76 Cephalexin 30 min urticaria 5 months Positive (+) Positive 19) JC F 49 Cephalexin Immediate Anaphylaxis 5 months Positive (++) Positive (SH & ST) 20) KL F - Cefazolin Immediate Anaphylaxis NM Positive (++) Positive (SH & ST)
Testing for beta lactam cross-reactivity SA pathology experience In Group 1(Penicillin allergic), 44 BAT were carried out for the cross reactants in all of the 11 patients, yielding 7 positive and 37 negative results. In Group 2 (Cephalosporin allergic), 34 BAT were carried out for the cross reactants in 6 of the patients, yielding 4 positive and 30 negative results When the BAT result was compared with available confirmed reactivity of the cross reactants as per the clinical confirmation, the following values were generated:
Clinical characteristics of venom allergic patients tested Sex Age Reaction sige JJA OD m 15 Grade 2 47 TB m 65 Grade 2 17 PW m ME m 60 Grade 1-2 1.6 GJ Jack Jumper Ant u DG m 34 Grade 1 8.4 VS f 63 Grade 2 36 JH m 16 Grade 2 30 JC m 27 Grade 2 2.2 GF f 64 Grade 2 >100 MO m 16 Grade 2 Honey Bee Venom Sex Age Reaction sige HBV MM m 52 Grade 2 On IT MC f 68 Grade 3 On IT 44 1.2 MK f 39 Grade 2 46 DW DL m 76 6.8
Current Status in our Lab Protocol now established at SA Pathology Ongoing critical review of method and results Adjunct in ongoing trials JJA Peanut Aravax, boiled peanut immunotherapy trial Cat allergy vaccine trial Drug allergy Antibiotics NSAIDs Anaesthetic agents Food allergy potentially useful as adjunctive tool to reduce requirement for challenge Novel/rare allergens (case reports)
Summary BAT is a very useful test in allergy diagnosis Differentiates between sensitisation and clinical allergy Caveats Standardised lab protocols Well designed experiments Allergen concentration/composition Reporting guidelines
Allergy research lab
Key References The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease. H. J. Hoffmann et al, Allergy 2015; 70:1393 1405. Steiner et al. Basophil reactivity as biomarker in Immediate Drug Hypersensitivity Reactions potential and limitations. Frontiers in Pharmacology June 2016