Quality Controls in Allergy Diagnosis Alistair Crockard Royal Hospitals Belfast Northern Ireland
Quality Controls in Allergy What do we want? Diagnosis What can be controlled? What can be achieved?
What do we want? 1. A process that ensures that: the results generated are correct
What do we want? 1. A process that ensures that: the results generated are correct QUALITY CONTROL
What do we want? 2. A process that ensures: the quality of results is consistent and in line with those determined for recognised reference material (standards)
What do we want? 2. A process that ensures: the quality of results is consistent and in line with those determined for recognised reference material (standards) QUALITY ASSESSMENT
What do we want? 3. A process that ensures: the correc test is carried out on the correct sample and the correct result, with the correct interpretation, is delivered to the correct person at the correc time.
What do we want? 3. A process that ensures: the correc test is carried out on the correct sample and the correct result, with the correct interpretation, is delivered to the correct person at the correc time. QUALITY ASSURANCE
What do we want? 4. Guidelines: CLSI Approved Guidelines I/LA20-4 (1997) Evaluation methods and analytical performance characteristics of immunology assays for human IgE antibodies of defined specificities 2 nd Edition due late 2008 www.clsi.org
What can be controlled? Steps in the Analytical Process
What can be controlled? Steps in the Analytical Process Blood Sample Assay Results Report correct patient correct sample correct request instrument reagents method assay performance validation of results interpretation
What can be controlled? outside and inside the laboratory Outside Instrumentation / Assay Assay Methodology Detection System Assay Sensitivity Reagents Preparation Purity Allergenicity Reports Clinical Interpretation
What can be controlled? outside and inside the laboratory Outside Instrumentation / Assay Assay Methodology Detection System Assay Sensitivity Reagents Preparation Purity Allergenicity Reports Clinical Interpretation Inside Assay Performance Precision Accuracy Reproducibility Quality Control Quality Assessment Result Interpretation
What can be achieved? Outside the Laboratory Instrumentation / Assay Methodology / Reagents Commercial Companies Extensive Quality Programmes
Quality Control procedures for components used in Specific IgE assay Serum Allergen Raw material Raw Sponge Selected control sera Antibody Activated Sponge Enzyme ImmunoCAP IgE Calibrator WHO reference preparation Conjugate Buffer Software Instrument Assay Development Solution aige ImmunoCAP Antibody Activated Sponge Wash Solution Stop Solution Buffer Substrate Raw Sponge Quality Control / methods and specifications
What can be achieved? Outside the Laboratory Reagents Heterogeneous starting material Variable extraction/purification/synthetic procedures Natural / recombinant allergens Material of variable allergenicity Standardisation problematic
What can be achieved? Outside the Laboratory Allergen Standardisation limited WHO International Reference Preparation 75/502 for human IgE generally adopted as industry standard WHO International Reference Preparations for birch pollen; dog hair/dander; HDM (D pteronyssinus); ragweed pollen; timothy grass pollen generally ignored as industry standard Units: kiu/l and ku A /l not equivalent
d1 e1 g6 t3
CREATE Project Consortium of interest groups: Clinical; Research; Industrial; Regulatory Task: Produce purified recombinant and natural allergens Evaluate recombinant major allergens as candidate certified reference materials Validate sandwich ELISAs for quantitation
Panel of 9 recombinant molecules studied 3 molecules identified which displayed good structural and immunological reactivity and biological potency: rbet v1; rphl p 5a; rder p 2 ELISAs verified rbet v 1 and rphl p 5a established as international reference standards by end 2008
What can be achieved? Inside the Laboratory Monitoring Assay Performance Implementing effective internal QC schemes Incorporating control sera, of defined specific IgE concentrations, into each assay run Plotting performance using Shewhart / Levy- Jennings plots
Shewhart Chart 100 KU/l 80 60 40 20 +3 sd +2 sd +1 sd Target value -1 sd -2 sd -3 sd 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Assay Run Allergen D1: Target Value = 49 ku/l
Step Change Bias Drift In Control
What can be achieved? Inside the Laboratory Monitoring Assay Performance Implementing effective internal QC schemes Incorporating control sera, of defined specific IgE concentrations, into each assay run Plotting performance using Shewhart / Levy- Jennings plots Applying Westgard rules to determine assay acceptability
Westgard Rules Used to define define specific performance limits for an assay Detect both random and systematic errors 6 commonly used rules 3 warning rules (1 2SD ; 2 2SD ; 4 1SD ) 3 mandatory (1 3SD ; R 4SD ; 10x)
Westgard Rules Violation of warning rules should trigger a review of test procedures, reagent performance and equipment calibration Violation of mandatory rules should result in the rejection of the results obtained with patients serum samples in that assay
Monthly IQC Peanut (F13) March 2008 Value 60.0 55.0 50.0 45.0 40.0 35.0 30.0 25.0 20.0 15.0 ASSAY: 03.03.08 04.03.08 05.03.08 06.03.08 07.03.08 10.03.08 11.03.08 12.03.08 13.03.08 peanut f13 (Immulite DPC) 14.03.08 18.03.08 19.03.08 Date kit kit expiry allergen allergen expiry Control A Control B Rule Violations CONTROL DATA COMMENTS lot no date lot no date Value SD Value SD 1 2S 1 3S 2 2S R 4S 4 1S 10 X and carry over 03.03.08 240 30.09.08 122 28.02.09 39.5 Control A Accept (GB) 04.03.08 240 30.09.08 122 28.02.09 41.6 1 Batch Accept (GB) 05.03.08 240 30.09.08 122 28.02.09 43.2 1 Mean SD CV% Accept (GB) 06.03.08 240 30.09.08 122 28.02.09 47.8 2 A 36.2 4.4 12.2 Accept (GB) 07.03.08 240 30.09.08 122 28.02.09 38.9 + Mean - Accept (GB) 10.03.08 240 30.09.08 122 28.02.09 40.0 Accept (GB) 11.03.08 240 30.09.08 122 28.02.09 36.7 Last 3 SD's Accept (GB) 12.03.08 240 30.09.08 122 28.02.09 38.1 27th Accept (GB) 13.03.08 240 30.09.08 122 28.02.09 36.0 28th Accept (GB) 14.03.08 240 30.09.08 122 28.02.09 38.9 29th Accept (GB) 18.03.08 240 30.09.08 122 28.02.09 41.5 Control B Accept (GB) 19.03.08 240 30.09.08 122 28.02.09 34.7 Batch Accept (GB) 20.03.08 240 30.09.08 122 28.02.09 42.9 1 Mean SD CV% Accept (GB) 21.03.08 240 30.09.08 122 28.02.09 41.4 1 Accept (GB) 26.03.08 240 30.09.08 122 28.02.09 40.4 + Mean - Accept (GB) 27.03.08 240 30.09.08 122 28.02.09 42.2 1 Accept (GB) 28.03.08 240 30.09.08 122 28.02.09 40.1 Last 3 SD's Accept (GB) 20.03.08 21.03.08 26.03.08 27.03.08 28.03.08
What can be achieved? Inside the Laboratory Particular difficulties in running allergen specific IgE IC schemes Limited availability of reference sera and commercial preparations Limited availability of in house serum samples of clinically relevant concentrations: CVs concentration dependent Resulting in the inability to run IQ for all allergenspecfic IgE assays Use data from representative allergen-specfic IgE assays
What can be achieved? Inside the Laboratory Monitoring Assay Performance Participating in external Quality Assessment schemes Comparative information about methods and analytical platforms Providing a measure of performance Commercial companies Distribution of sera of defined concentrations Internet-based service and support Independent providers Distribution of sera of defined concentrations Case histories / interpretative reporting
What can be achieved? Inside the Laboratory Monitoring Assay Performance Participating in external Quality Assessment schemes Comparative information about methods and analytical platforms Providing a measure of performance Commercial companies Distribution of sera of defined concentrations Internet-based service and support Independent providers Distribution of sera of defined concentrations Case histories / interpretative reporting
What can be achieved? Inside the Laboratory Monitoring Assay Performance Participating in external Quality Assessment schemes Comparative information about methods and analytical platforms Providing a measure of performance Commercial companies Distribution of sera of defined concentrations Internet-based service, support and QC programmes Phadia LabCommunity ; Siemens Real Time Solutions Independent providers Distribution of sera of defined concentrations Case histories / interpretative reporting
What can be achieved? Inside the Laboratory Monitoring Assay Performance Participating in external Quality Assessment schemes Comparative information about methods and analytical platforms Providing a measure of performance Commercial companies Distribution of sera of defined concentrations Internet-based service, support and QC programmes Phadia LabCommunity ; Siemens Real Time Solutions Independent providers Distribution of sera of defined concentrations Case histories / interpretative reporting
www.skml.nl Single donor samples with case histories Medical history Physical examination Physiological investigations Allergy investigations (SPT; challenge tests) Conclusion / Diagnosis
Sample 2008-04 Allergic rhinitis IgE sensitisation to HDM Grass pollen Animal dander Latex IgE
Sample 2008-04 Allergic rhinitis IgE sensitisation to HDM Grass pollen Animal dander Latex e1
Sample 2008-04 Allergic rhinitis IgE sensitisation to HDM Grass pollen Animal dander Latex e3
Sample 2008-04 Allergic rhinitis IgE sensitisation to HDM Grass pollen Animal dander Latex k82
www.ukneqas.org.uk www.immqas.org.uk Schemes IgE Allergen-specific IgE; 15 common allergens Single donor samples Performance assessment of quantitative responses and consensus of results by grades
Web-based educational tool Allows individuals to practice clinical and scientific interpretation skills on virtual patient results Navigate through screens of test results and medical information Suggest tests and investigations; draw conclusions Compare with correct answers Award certificate of participation
Cellular Tests
Cellular Tests Basophil Activation Allergen CD63+ CD203c+ Activated Basophil Anti-IgE
Basophil Activation Tests Standardisation Protocols Technical Aspects Interpretation
EuroBAT Project Initiative to establish the diagnostic validity of BAT in clinical scenarios where current tests are: Unreliable Difficult to perform Unavailable
EuroBAT Project Undertake multicentre studies to validate Protocols Reagents Commercial kits Produce position papers / guidelines Develop European standard reference protocols Maintain a website www.allergyflow.org
EuroBAT Working Groups Technical Issues Venom Food Autoimmune Urticaria Drugs Betalactams NSAIDS Muscle Relaxants
Quality Controls in Allergy What do we want? Diagnosis What can be controlled? What can be achieved?
Quality Controls in Allergy What do we want? Diagnosis What can be controlled? What can be achieved? What more to do?
Future Challenges Cellular Assays Micro Array Assays
Future Challenges Recombinant Allergens EMBO 2004
Future Challenges Recombinant Allergens Recombinant Antibodies I. Braren et al Clin Chem 2007 53: 837 EMBO 2004
Future Challenges Near Patient Testing
Patient Responses Genetic Diversity
Patient Responses Genetic Diversity 50 (something) year old atopic male Occasional rhinitis Specific IgE HDM: Positive 6 ku/l SPT HDM: Positive 4mm wheal Basotest HDM: Positive ImmunoCAP TM Rapid: NEGATIVE
Thank you for your attention