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Outline
Background topics that surround quality discussions - NRL latex allergy & NRL allergens - Attitude & awareness of general public - Standardisation news, discussions over further developments

FITkit technology - proposed latex industry quality standard

Analysis and proposals for future

Hardi Tamm, Icosagen AS 24 September, 2010 Kamunting

Long list of products containing latex

Latex can cause allergy

Over 40 000 different consumer products, up to 400 used routinely in Health Care Over 40 billion pairs of medical and surgical gloves used each year Large number of products packed with NRL materials

Why?
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Latex of the Para Rubber Tree

(Hevea brasiliensis)

Allergens in rubber products

Raw latex contains at least 200 different proteins, 50 - 60 of which have been named potentially allergenic (binding human IgE) The WHO/IUIS Allergen Nomenclature Sub-Committee currently (March 2010 ) lists 13 NRL allergens characterized at the molecular level. level Only 4 of them are clinically relevant (major allergens)

34% polyisoprene (rubber) 1% proteins 0.4% fatty acids 1% resins 0.6% ash 1.4% sugar 60.0% water

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Latex allergens nomenclature (2010)

Hev b 1 (Rubber elongation factor; REF) Hev b 2 (Endoglucanase) Hev b 3 (23-27 kDa rubber particle protein) Hev b 4 (Microhelix protein complex) Hev b 5 (Acidic 16 kDa protein) Hev b 6.01 (Prohevein, hevein preprotein) Hev b 6.02 (Hevein, mature hevein) Hev b 7 (Patatin-like Hevea protein) (Patatin like Hev b 8 (Hevea profilin) Hev b 9 (Hevea enolase) Hev b 10 (Manganese superoxide dismutase) Hev b 11 (Hevea endochitinase) Hev b 12 Hevea Lipid transfer protein (LTP) Hev b 13 (Esterase) Early nodulin specific protein
brasiliensis)

Latex allergy is it a problem?

Allergy prevalence based on Skin Prick testing Cross-reactivity with latex
More known cross-reactions:

Children with spina bifida 32 - 51 % Health care workers 3 - 11 % Operating room personnel 8 - 10 % General population <1%

Latex fruit Latex-fruit syndrome Cross-reactions with pollens Cross-reactions with moulds

Look at http://www.allergen.org (Home > Plantae Magnoliopsida > Malpighiales > Hevea

Some socks, as well many textile products contain natural latex threads

The trends and risks, the attitudes and awereness of the general population

Latex allergy seems to be in a continuous rate decline among healthcare workers (HCW); Calls for better labelling & information towards rubber products There is a risk of being too ignorant in circumstances where we believe the (latex allergy) "epidemy" to be over; Proposals apply similar requirements for the medical & the consumer products Increasing control of the synthetic products possible NRL content Continuous risk of sensitisation among the general public Missing control over the allergenicity of consumer products by regulatory bodies Very low and hazy awareness among general public Available information is usually insufficient
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Eczema
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Can exposure to latex allergens be lowered?

Actions taken to reduce latex sensitisation

Specific procedures in the end of production lines Replacing or coating latex with synthetic polymers Handling of raw latex to reduce allergens Latex free environment Choosing low allergen and/or powder free latex gloves using gloves from accurately tested batches only Improvement of technology to reduce allergen content in the product Proper QC/QA in production
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Yes, by controlling the levels of extractable allergens in manufactured products Ideally, no allergens should be quantifiable in latex products Problem: it has not been possible to establish any safety limits.

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Current testing methods 1(3)

Total protein measurement Antigenic protein measurement
ELISA for Latex Antigenic Proteins LEAP assay or ASTM D6499

What we need a proper method to quantify specific allergens

Lowry/Modified Lowry Method ASTM D5712, EN 455-3:2006 Amino Acid Analysis

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Criticism
By measuring the total protein level in the latex product extract no direct information can not be obtained on whether or not the mixture contains allergens Antigenic proteins test result does not directly correlate with the allergen content of the end product because It measures antigenic proteins not allergens.

Current testing methods 2(3)

(Available methods to measure allergenic proteins in latex products extracts)

Skin prick tests in voluntary latex allergic patients IgE - immunoblotting IgE ELISA-inhibition methods - RAST/ ELISA Inhibition g / Assays (IgE) Capture EIA methods with monoclonal antibodies to specific allergenic proteins

Why to measure non-relevant proteins?

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Current testing methods 3(3)

Immunologic quantification of allergen content

FITkit
Four allergens were chosen for FITkit assays that should be measured in medical gloves
- two major ones in adult allergies (Hev b 5, Hev b 6.02) - two major ones in children's allergies (Hev b 3, Hev b 1)

FITkit technology; ASTM D7427-08, EN 455-3:2006 FITkit

The first specific tests for identifying and quantifying individual NRL allergens Direct measurement of relevant allergens individually High sensitivity Consistent and reliable results Convenience and short assay time (2 hours) No interference from other substances

These four allergens can be present in NRL products after the g p p manufacturing process According to literature, the sum of these four allergens shows a high correlation to total allergenicity measured by SPT or ELISA Inhibition Assay

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Key components of FITkit

Capture-EIAs for the measurement on latex allergens

Purified and characterized proteins (known at the molecular level) recombinant allergens or purified from natural sources monoclonal antibodies with unique specificities

Enzyme conjugated anti-Hev b 5

- monoclonal antibodies are both scientifically and technically proven to be superior test reagents

Hev b 5

Anti-Hev b 5

Monoclonal antibodies and recombinant allergens required

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Standardization status

Allergenicity and disputes over the allergen content limit

for the Numerical values to determine any safety levels of gloves and other NRL products in terms of latex allergens had not been established before 2007 In CEN a term ALARP (As Low As Reasonably Practicable) has been adopted There are products on the market in which the allergens level is below the quantitation limit when measured with the most sensitive methods (FITkit) Manufacturing processes has been improved; Desire to find 100% safety

European standard EN455-3:2006

Modified Lowry (normative), Annex A Immunological methods, including capture enzyme immunoassays (informative) like FITkit, Annex B Amino acid analysis by HPLC (informative), Annex C

ASTM

D7427-08

Standard

Test

Method

Immunological Measurement of Four Principal Allergenic Proteins (Hev b 1, 3, 5 and 6.02) in Natural Rubber and its Products derived from Latex
(http://www.astm.org/Standards/D7427.htm).

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Assessing the level of allergenicity of NRL products

Main values of the new classification

Uses accurate allergen detection solution; Makes it possible to differentiate low allergen products from others; It encourages the industry to continue improvement of production technology; It is applicable for different interest groups, from standardization bodies to general public; g p ; It is suitable for different products, from gloves to consumer products; It is not in conflict with any already accepted solutions; Allows the assessment of products which are unable to meet the 0.15 g/g threshold and monitors the quality improvement of products over time; Leads us to safer usage of NRL
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Very low allergenicity (unquantifiable, i.e. sum of the four allergens < 0.03 g/g) Low allergenicity (0.03 0.14 g/g) Borderline allergenicity (0.15-0.29 g/g) Moderate allergenicity (0.30-1.14 g/g) High allergenicity (>1.15 g/g) g g y(
(FITkit detection)

Finnish National Agency for Medicines Glove Study 2005 Palosuo, T. et al., Latex allergy: The sum quantity of four major allergens shows the allergenic potential of medical gloves Allergy 62, 781-786 (2007)

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Take home message

All proteins are immunogenes but only few are allergens; We can not ignore any type of allergy; Not only gloves but many consumer products contain high amount of latex allergens similar requirements help to understand; FITkit meets the requirements of the appropriate technology; New allergenicity classification makes latex products assessment g y p easy: When the sum of four allergens is below 0.15 g/g the latex product is more likely safe Products over 1.15 ug/g having high allergenicity potential Quality of NRL and the adequate data supports the safe use of NRL
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Thank you!

Icosagen Group www.icosagen.com www icosagen com info@icosagen.com