National Measurement

Institute” and the Allergen

Testing Special Interest

Group

March 2013

National Measurement Institute Port Melbourne, Australia

National Measurement Institute

The National Measurement Institute is Australia’s peak

measurement organisation, responsible for the national

measurement infrastructure (biological, chemical, legal,

physical and trade measurement) and for maintaining

Australia’s primary standards of measurement

We are a division within the

Department of Innovation, Industry, Climate Change

Science and Research and Tertiary Education

(DIICCSRTE)

http://www.measurement.gov.au

National Measurement Institute

Microbiology Laboratory

Allergen Laboratory

Budapest Treaty Collection (Patent organisms)

Food Chemical Analysis

Environmental Analysis

Sports Drug Testing

Illicit Drug analysis

Time (Australia’s Atomic Clocks)

Physical Metrology – Mass, Length, Frequency

Legal Metrology – Patent Approvals

Reference Material Production

Proficiency Testing Schemes

Trade Measurement (Fuel pumps and Commercial scales)

NMI – Allergen Background National Measurement Institute – 20 years of food allergen analysis

Chromatography expertise within NMI – GC, LC, CE, MS, Bioanalyzer

Current Food Allergen analysis:

Gluten, Peanut, Sesame, Soy, Egg, Milk (Casein, Beta lactoglobulin),

Crustaceans, Almonds, Hazelnuts, Walnut, Pistachio, Macadamia, Cashew

PCR experience - GMO, food pathogens, digital PCR and some food

allergens

Implemented an automated ELISA analysis system

– Hamilton’s Micro Star liquid handling robot

Developing Chromatographic methods for allergen protein detection

– Mass accurate, Waters G2 Q-TOF

Allergen Activities – Education and

Participation Allergen Testing Special Interest Group

Australian Standards committee on Allergens

Proficiency rounds and materials

NATA assessors for allergen labs

Confirmation techniques

TIA (Technical Infrastructure Alliance) proposal

Technical workshops : – NMI Allergen Measurement Workshops – July 2009, in 3 states, 2 workshops, 3 evening seminars over 100

attendees from Industry – targeting SME

– Technical workshops in 2011 Brisbane as part of GFAC

Proficiency Testing

Proficiency testing is a means of assessing the ability of

laboratories to competently perform specific tests and

measurements

Proficiency testing can setup so the samples

are tested:

• internally (results compared within the laboratory)

• externally (results compared between different

laboratories)

Proficiency Testing & Quality

Control Materials

Benefits:

• Satisfy accreditation requirements

• Verify methods and procedures

• Satisfy regulators

• Identify problems

• Enhance accuracy

• Monitor analysis

• Compare with peers

• Demonstrate and Build confidence

Quality Control Materials

Used with every kit run

Provide:

– Within run control checks – assay drift

– Between run variations

– Batch to batch kit variations

– Comparison between kits

– Comparison between labs

– Measurement uncertainty data

Proficiency Round on Gluten in Custard

Powder AQA 10-21

Samples prepared by NMI PT section

Gluten in custard powder selected as a representative matrix

11 labs

Custard powder obtained for a commercial supplier

– Gluten containing product

– gluten free product

sieved and mixed

packaged into 100 g portions

Gluten test kits from different manufacturers can give very different results

All distinguish between gluten-containing and gluten-free, but quantitative results from different kits are not comparable

Proficiency Round on Sesame in Chocolate

Cake AQA 12-10

Real world (not incurred) sample of cake mix containing low level

sesame protein

recalled product with history of reactions in allergic individuals

7 labs participating

All correctly identified positive and negative samples

MU quoted ranged from 7.7 to 290%

Included re-run of gluten custard powder proficiency samples

11 labs participating – nine submitted resultS

Much better co-relation of results in follow up round

Allergen Quality Control Material

What does this “simple” reference material give us?

An inexpensive regular use material

Comparison between labs and kits

An ability to track kit variations and variations between different kit manufactured kits

Option to tailor-make a material with desired target level by mixing target containing and target free materials.

A real “incurred sample”

Small dry particles for stability

Shelf stable storage

Supporting quality assurance that the daily ELISA run is performing appropriately

Food Allergy in Australia

Australia and New Zealand among the highest prevalence of allergic disorders in the developed world

Estimated 4.1 million Australians have at least one allergy (19.6%)

Hospital admission x2 in last 10yrs

Increase of 5 fold for 0-4 year olds

Peanut allergy x2 in 5yr period

Financial cost of Allergies 7.8 billion in 2007

Data published in October Issue of Journal of Allergy and Clinical Immunology

Precautionary Labeling

May contain occasional nut

May contain peanuts, nuts and other allergens not listed on the label

Allergy information: Baked in a facility that uses dairy

May contain unknowns substances

May contain traces of vitamins and water (snack bar)

Coconut milk is gluten free and therefore is suitable for those with cow’s milk allergy

Global differences and definitions

Australia and New Zealand – Coconut not generally considered a tree nut

EU – following tree nuts defined – Almonds, Brazil nuts, Cashews, Hazelnuts, Pecan nut, Pistachio nut, Walnuts, Macadamia or Queensland Nut

USA : tree nuts defined – Almond, Beech, Brazil nut, Butternut, Cashew, Chestnut, Chinquapin, Coconut, Filbert/hazelnut, Ginko nut, Hickory, nut, Lichee nut, Macadamia nut/ Bush nut, Pecan, Pine nut/ Pinon nut, Pistachio, Sheanut, Walnut

Canada - almonds, Brazil nuts, cashews, hazelnuts [filberts], macadamia nuts, pecans, pinenuts, pistachios, walnuts

Peanuts which are legumes - earthnuts, ground nuts, goober peas, monkey nuts, pygmy nuts and pig nuts

Global differences and definitions

EU Cereals defined: Wheat, Oats, Barley, Rye, Kamut, Spelt, Triticale

USA - The term "wheat" in regulation refers to any species in the genus Triticum…. so wheat would include semolina, Einkorn, emmer in addition to EU list

Japan – Mandatory and Recommended list - Buckwheat

Codex Alimentarius etc

Driven by available analysis tools

Food Allergen Management in

Australia

FSANZ mandatory labelling 2002

Advisory or precautionary

statements

Allergen Bureau – Info resource

GMP – Hygiene control

Recalls

Raw ingredient and final product

testing

Industry Tool Kit

AFGC – Allergen Management and

Labelling Guide

Allergen bureau

Food Industry Product Information Form (PIF)

The VITAL tool

Analysis tools

Metrology or Measurements

Physical Measurements – Amount (length, time, mass)

Chemical Measurements – Amount (ppm) Type of substance (Benzene

Biological Measurements – Amount (mg/kg)

Allergen protein (Ara h1 peanut etc)

Activity / Allergenicity (digestibility, stability,

heat resistance, irraditated,

active sites available ,

modified or concealed)

Accuracy & Precision

Accuracy is the degree of

closeness of a measured

or calculated quantity to

its actual (true) value

Precision, also called

reproducibility or

repeatability, the degree

to which further

measurements or

calculations show the

same or similar results

Accuracy & Precision

Accuracy

Accuracy in Food Allergen analysis is very difficult to

estimate due to: • difficultly in defining the test parameter, target or measurand – gluten,

gliadin, ethanol/water extractable component, penta peptide, G12

sequence

• Modifications to target protein

• inherent variations in biological systems

• lack of reference standards

• Inhibitory or matrix impacts of food – salt, pH etc

• Sampling variation due to uneven distribution of allergen

Precision

Easier to estimate when compared to accuracy of food

biological measurements

Referred to as reproducibility or repeatability

The reproducibility of results is what is often used to

estimate measurement uncertainty for Biological

measurements

Measurement Uncertainty

(MU)

MU Introduction “A parameter associated with the result of a measurement that

characterises the dispersion of the values that could reasonably

be attributed to the measurand.”1,2

1 “International vocabulary of basic and general terms in metrology” (VIM), (1993), 2nd ed., ISO, Geneva, Switzerland. 2 “ISO/TS 19036 (ISO GUM).

Biological MU

• A “top-down” or “global” approach is preferred world wide

for Biological MU

• Based on the standard reproducibility of the final

measurement process

• Based on the overall variability of the analytical process

• Estimation of the laboratories analytical method as a

whole, ignoring individual analytical components

Decision Making

(a) When compliance decisions are clear, then only rough estimates of MU are needed.

(b) When measured results are close to decision points, good estimates of MU and MU values will improve decision making.

(c) Where false positive decisions are undesirable, underestimation of MU can lead to inappropriate decisions.

(d) In some cases the risk of compliance/non-compliance can be shared. Careful consideration would need to be given to the level of MU and the reliability of the estimation.

MU from Within-Lab Derived Data

• Repeatability (common approach)

• Stable, consistent product

• Multiple runs on different days

• Different operators

• Different kit batches

• Upper and lower temperature range in Lab

MU from External Proficiency

Testing Data

• Use already existing data

• Represents a long term MU calculations

• Must be same methods

• Acceptable range for International PT +/- 50%

• Calculated MU +/- 30%

Measurement Uncertainty for

Biologists – Course run by NMI

This one-day course explains the general principles of estimating measurement uncertainty in

quantitative biological analysis. Interactive workshops with case studies based on real

analytical problems are used to reinforce these principles.

Objectives

– The course provides participants with the knowledge and skills to:

– understand the principles involved in measurement uncertainty and traceability

– apply uncertainty concepts when developing and conducting routine analysis

– make ISO/IEC 17025 compliant estimates of measurement uncertainty

Outline

– The topics covered include:

– uncertainty definitions and concepts

– sources of uncertainty in calibrants

– uncertainty from the measurement process

– combining uncertainties

– calculating and reporting expanded uncertainties

– worked examples

AT-SIG: Target audience

Allergen Bureau Membership

Testing Laboratories

Accreditation bodies

Regulators

Industry Bodies

Consumer Advocates

Kit Manufacturer or Distributor

AT-SIG: Group Aim

Improve food allergen analysis

Share challenges and issues - Address common or reoccurring issues

Keep labs talking – particularly on overlapping issues

Provide a forum for formal and informal networking

Encourage input from all parties

Forum to present emerging technology applicable to food allergen analysis

Assist Laboratory accreditation

Guide – Proficiency, Reference Materials and Confirmation Methods

Keep up to date with International (and local) Allergen Activities

AT-SIG: Activities

First meeting held: October 2010 @ NMI Port Melbourne

Second meeting held: June 2011 @ NMI Port Melbourne

Third meeting held: April 2012 @ NMI Port Melbourne

25-30 participants per meeting

AT-SIG - Meeting Topics

Case studies

Proficiency Testing

Laboratory Accreditation for Allergens

Reporting Issues

Up coming Food Allergen events (Local and International)

Reference Materials

New technology

Reporting document

Input from Stakeholders: - 'greater than' results - all but useless for risk assessment purposes - lack of clarity around 'less than' results i.e. less than the LOD, or detected but less than the LOQ - lack of clarity around the whether the kit is calibrated/reporting against the allergenic protein, total protein or whole food - variability of results, including detections with different kits - significant in survey work where a number of labs may be involved

To address: - Reporting results below LOQ but above LOD - Numbers vs ranges

- Analyte or measurand – target - Units (ppm,mg/kg)

- Assumptions or exclusions Swabs - Sampling & sample size

- Known or suspected cross-reactivities - Measurement Uncertainty

- What to do with unexpected / disputed results - etc

AT-SIG: What have we learnt?

Analysts are keen to discuss issues (reasonably open) – despite competing commercial interests

Labs keen to be there (or scared not to be)

Discussion time as important as presentation – in fact presentation are often the background information leading into the discussions

We (Labs) often share the identical problems, sample, matrix, disputed results etc

Confirmation methods are important and needed

Benefits and Outcomes

Directly linked into industry issues – valuable information and shared experiences

Forum for immerging issues that brings experts into a single space - a resource to address immediate and longer term food allergen concerns

Aims to be conscious of but not completely restricted by confidentiality issues

Informal opportunities to raise concerns

Opportunity to be involved in shared research and development of allergen relevant protocols and documents

Drives and guides future Allergen activities – PT, Reference Materials and Confirmation Methods

Benefits and Outcomes

Precompetitive environment to discuss critical issues

Mentoring of labs with less opportunity to see challenging of issues

Proficiency program for participants (and others)

Reporting document

and potentially other guidance documents

Links participants into National and International activities

Future of AT-SIG

1 meeting per year

Communication – web space

Continue to seek relevant participants

Keep up to date with International developments

through network contacts

Email: dean.clarke@measurement.gov.au for more

information

THANK YOU

National Measurement Institute

1/153 Bertie Street

Port Melbourne

Australia

Phone: + 61 3 9644 4888

Email: dean.clarke@measurement.gov.au