Medical gloves made from natural rubber latex -- Determination of water-extractable protein using the modified Lowry method

ISO 12243:2003 specifies a method for the determination of the amount of water-extractable protein in natural rubber (NR) gloves for medical use. The method is potentially suitable for the determination of extractable protein in other articles made from NR latex; however the extraction procedures and times have not been validated and will vary with the type of article to be tested. Other methods for the determination of specific proteins in medical gloves exist (these are described in an annex) but they are not of general applicability. This International Standard is concerned solely with the method of assay. It is not concerned with sampling nor does it purport to address the safety implications of the values obtained or requirements for labelling.

Gants médicaux à base de latex de caoutchouc naturel -- Détermination des protéines extractibles par l'eau par la méthode modifiée de Lowry

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Status
Published
Publication Date
22-Sep-2003
Current Stage
6060 - International Standard published
Start Date
03-Jul-2003
Completion Date
23-Sep-2003
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INTERNATIONAL ISO
STANDARD 12243
First edition
2003-10-01
Medical gloves made from natural rubber
latex — Determination of
water-extractable protein using
the modified Lowry method
Gants médicaux à base de latex de caoutchouc naturel —
Détermination des protéines extractibles par l'eau par la méthode
modifiée de Lowry
Reference number
ISO 12243:2003(E)
ISO 2003
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ISO 12243:2003(E)
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© ISO 2003

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ii © ISO 2003 — All rights reserved
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ISO 12243:2003(E)
Contents Page

Foreword............................................................................................................................................................ iv

Introduction ........................................................................................................................................................ v

1 Scope...................................................................................................................................................... 1

2 Normative references ........................................................................................................................... 1

3 Principle ................................................................................................................................................. 1

4 Terms and definitions........................................................................................................................... 2

5 Apparatus............................................................................................................................................... 2

6 Reagents ................................................................................................................................................ 3

7 Procedure............................................................................................................................................... 4

8 Calculation of results............................................................................................................................ 7

9 Precision ................................................................................................................................................ 8

10 Test report.............................................................................................................................................. 9

Annex A (normative) Verification.................................................................................................................... 11

Annex B (normative) Protein adsorption on polypropylene and polyethylene tubes............................... 13

Annex C (informative) Alternative methods of analysis ............................................................................... 14

Annex D (informative) Background subtraction............................................................................................ 15

Bibliography ..................................................................................................................................................... 17

© ISO 2003 — All rights reserved iii
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ISO 12243:2003(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 12243 was prepared by Technical Committee ISO/TC 45, Rubber and rubber products, Subcommittee

SC 3, Raw materials (including latex) for use in the rubber industry.
iv © ISO 2003 — All rights reserved
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ISO 12243:2003(E)
Introduction

There have been problems of allergic reactions experienced by some users of medical gloves manufactured

from natural rubber latex. ISO 12243 specifies a method for the determination of the water-extractable protein

in such gloves.
© ISO 2003 — All rights reserved v
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INTERNATIONAL STANDARD ISO 12243:2003(E)
Medical gloves made from natural rubber latex — Determination
of water-extractable protein using the modified Lowry method

WARNING — Persons using this International Standard should be familiar with normal laboratory

practice. This standard does not purport to address all of the safety problems, if any, associated with

its use. It is the responsibility of the user to establish appropriate safety and health practices and to

ensure compliance with any national regulatory conditions.
1 Scope

This International Standard specifies a method for the determination of the amount of water-extractable

protein in natural rubber (NR) gloves for medical use. The method is potentially suitable for the determination

of extractable protein in other articles made from NR latex; however the extraction procedures and times have

not been validated and will vary with the type of article to be tested. Other methods for the determination of

specific proteins in medical gloves exist (see Annex C) but they are not of general applicability.

This International Standard is concerned solely with the method of assay. It is not concerned with sampling

nor does it purport to address the safety implications of the values obtained or requirements for labelling.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated

references, only the edition cited applies. For undated references, the latest edition of the referenced

document (including any amendments) applies.
ISO 10282:2002, Single-use sterile rubber surgical gloves — Specification

ISO 11193-1:2002, Single-use medical examination gloves — Part 1: Specification for gloves made from

rubber latex or rubber solution
3 Principle

Water-soluble proteins are extracted into a buffer solution and then precipitated to concentrate them and

separate them from other water-soluble substances which may interfere with the determination

(see Annexes A and D). The precipitated protein is redissolved and quantified colorimetrically by the modified

Lowry method using a protein standard (for a general review of the method, see reference [1] in the

Bibliography).
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ISO 12243:2003(E)
4 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
4.1
concentration factor

extent to which a protein extract is concentrated by precipitation followed by redissolution in a smaller volume

of sodium hydroxide solution
3 3

NOTE Thus if the protein in 4 cm of solution is precipitated and redissolved in 0,8 cm , then the concentration factor

F would be 4/0,8 (= 5).
4.2
protein

proteins and protein-like substances (e.g. polypeptides) occurring in articles made from NR latex and which

are extractable with water
4.3
modified Lowry method

modification of the original Lowry assay method, which involves the precipitation and isolation of the proteins

to reduce the level of other water-extractable substances that may interfere in the determination

5 Apparatus

Unless otherwise stated, all laboratory equipment (i.e. flasks, tubes, etc.) shall be made of polypropylene or

polyethylene.

NOTE Polypropylene or polyethylene equipment is specified rather than glass to minimize surface adsorption.

A method for the determination of protein-binding capacity is described in Annex B.

5.1 Protein-free gloves, made from synthetic rubber latex or plastic and that are free of powder and other

materials capable of being transferred to the test samples or extractant solutions.

5.2 Centrifuge, capable of reaching not less than 60 000 m/s (6 000 × g).

NOTE A refrigerated centrifuge is preferred as it is possible for the temperature to rise considerably when

centrifugation is carried out for prolonged periods.
3 3 3 3 3

5.3 Centrifuge tubes, capacity 200 cm , 50 cm , 10 cm , 2 cm and 1,5 cm , made of polypropylene or

polyethylene (if available) with a low protein-binding capacity.
5.4 Conical flasks, capacity 250 cm .
5.5 Micropipettes.
5.6 Test tube shaker, operating at between 3 Hz and 6 Hz.
5.7 Vortex mixer or ultrasonic bath.

5.8 Disposable filter, with a low protein-binding capacity and a pore size of 0,45 µm or less.

5.9 Clamps, for sealing gloves watertight during extraction. Pairs of aluminium bars lined with foam rubber

which can be screwed together, or 170-mm-long plastic clips as used for haemodialysis, are suggested.

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ISO 12243:2003(E)
5.10 Spectrophotometric equipment.

5.10.1 Spectrophotometer, with disposable polystyrene cuvettes (quartz cuvettes may be used but require

careful cleaning).

5.10.2 Microplate reader, with flat-bottom polystyrene microtitre plates having 96 wells of 0,25 cm to

0,5 cm capacity.

NOTE Wells with a capacity of 0,5 cm are preferred. Wells with a smaller capacity may be used but will reduce the

sensitivity of the assay.
5.11 Balance, accurate to 0,000 1 g.
6 Reagents

During the assay, use only reagents of recognized analytical grade and distilled or deionized water.

6.1 Dye solution: Bromophenol blue, sodium salt, prepared by dissolving 0,1 g of bromophenol blue in 1 l

of water. Discard the solution after four weeks.

6.2 Extractant solution: A buffer solution capable of maintaining pH 7,4 ± 0,4 throughout the extraction.

NOTE 1 Suitable buffers include phosphate buffer saline (PBS) solution (0,01 mol/l) and N-tris-(hydroxymethyl)-methyl-

2-amino-ethanesulfonic acid hemisodium salt (TES) solution (0,1 mol/l). The PBS buffer is prepared by dissolving a PBS

tablet in distilled water in accordance with the manufacturer’s instructions. In the event that, at the conclusion of the

extraction, pH 7,4 ± 0,4 is not achieved, it would be necessary to use a more concentrated buffer solution. The TES

solution is prepared by dissolving 24 g of TES in 500 cm of water and making the volume up to 1 l.

NOTE 2 PBS tablets and TES are widely available.
6.3 Modified Lowry protein assay reagents

6.3.1 Reagent A: Alkaline copper citrate, prepared fresh daily by mixing 10 parts of reagent C with 0,2 parts

of reagent D.

Alkaline copper tartrate is also considered to be suitable. It shall also be prepared fresh daily. The material

available in kits can contain undeclared preservatives which may affect the determination.

3 3

6.3.2 Reagent B: Dilute Folin reagent prepared by diluting 72 cm of 2 N Folin reagent with 28 cm of water.

NOTE 2 N Folin reagent is available commercially. It can, for example, be obtained from Sigma Chemical Co.

(Catalogue No. F 9252), Box 14508, St Louis, MO 63178, USA. The concentration of some commercial Folin reagents

may not be 2 N.
6.3.3 Reagent C: A solution of 6 g of sodium carbonate in 100 cm of water.

6.3.4 Reagent D: A solution containing 1,5 g of copper sulfate and 3 g of sodium citrate in 100 cm of water.

6.3.5 Sodium hydroxide solution, c(NaOH) = 0,2 mol/l.

6.3.6 Sodium deoxycholate (DOC) solution, prepared by dissolving 0,15 g of sodium deoxycholate in

water and diluting with water to 100 cm . Store the solution in a refrigerator, discarding it after 4 weeks.

6.3.7 Trichloroacetic acid (TCA) solution, prepared by diluting 72 g of trichloroacetic acid to 100 cm with

water and mixing thoroughly. Store the solution in a refrigerator. The solution is stable over a long period.

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ISO 12243:2003(E)

6.3.8 Phosphotungstic acid (PTA) solution, prepared by diluting 72 g of phosphotungstic acid to 100 cm

with water and mixing thoroughly. Store the solution in a refrigerator, discarding it after 4 weeks.

It may be convenient to premix the TCA and PTA solutions in equal volumes and to add them simultaneously

in 7.4.2. Such a mixture shall be prepared daily in the absence of data on its storage life.

6.4 Ovalbumin protein stock solution.

Use ovalbumin prepared by ammonium sulfate fractionation and repeated crystallization at pH 4,5 such as

Sigma A 5503 from Sigma Chemical Co., Box 14508, St Louis, MO 63178, USA.

Prepare a solution of 100 mg of ovalbumin in 100 cm of the preferred extractant (6.2) to give a concentration

of 1 mg/cm . Filter the solution through a low-protein-binding filter of 0,45 µm or smaller pore size and

determine the absorbance at 280 nm using a UV spectrophotometer with a 1 cm path length cuvette and

employing extractant solution (6.2) as a blank. Divide the absorbance by 0,64 to obtain the precise

concentration of the ovalbumin stock solution. The solution is stable for 2 days when stored at a temperature

of not more than 7 °C or for 2 months frozen at −10 °C. Thawing requires heating to 45 °C for 15 min.

NOTE The length of time under refrigeration is cumulative. In order to avoid repeated thawing and freezing, it is

recommended that the stock solution be stored as aliquot portions each sufficient for the preparation of a single calibration

curve or for use in the verification procedure (see Annex A).
7 Procedure
7.1 Principle

The procedure involves the extraction of a whole glove followed by purification and concentration of the

extract. The concentration of protein in the extract is determined by reference to a standard calibration curve

prepared using dilutions of the protein stock solution (6.4 and 7.3) which has been concentrated in the same

manner. The analytical technique of the analyst must previously have been verified as described in Annex A.

The extraction is run in triplicate using three gloves or pairs of gloves from a given lot; the purification and

concentration of each extract and the subsequent determination are run singly.
7.2 Extraction procedure
7.2.1 General

The entire surface of the glove shall be exposed to the extractant at 25 °C ± 5 °C for a period of

120 min ± 5 min. Two extraction procedures are permitted, the so-called “cut-glove” procedure and also the

“glove-in-glove” procedure. The procedure used shall be noted in the test report and all samples in a given

series shall be extracted by the same procedure. The extraction shall be carried out in triplicate and single

determinations run on each extract.

Use protein-free gloves (5.1) to handle the glove samples used for the extraction.

NOTE The frequency of sampling and left- or right-handedness of gloves are outside the scope of this document.

7.2.2 Procedure A — Cut-glove procedure

7.2.2.1 Record the mass of the glove (m) to an accuracy of not less than 0,001 g.

1) The precise value of the extinction coefficient of ovalbumin is subject to confirmation.

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ISO 12243:2003(E)

7.2.2.2 Cut the glove along the periphery. To facilitate the extraction, it is permissible to cut the glove into

smaller pieces (but see 7.2.2.3).

7.2.2.3 If the result is to be reported in micrograms per unit area of the glove (e.g. µg/dm ), determine the

surface area of the glove as follows:

Cut a rectangular piece from the back of the glove of about 0,5 dm by 0,5 dm and measure its dimensions

accurately. Calculate the area A .
Determine the mass (m ) of the rectangular piece to the nearest 0,001 g.
The total surface area A of both sides of the glove is given by A = 2A × m/m .
1 p
7.2.2.4 Transfer all the pieces of the glove to a suitable conical flask (5.4).

7.2.2.5 Add accurately a volume V of extractant (6.2). The total volume V of extractant used shall be

3 3
between 10 cm and 15 cm per gram of glove and sufficient to cover the pieces.

7.2.2.6 Extract the test sample at 25 °C ± 5 °C for 120 min ± 5 min, shaking for 15 s initially and

thereafter at intervals not greater than 30 min. If practical, continuous slow shaking is desirable.

7.2.2.7 Decant off the extract and remove any particulate matter by centrifuging at not less than

20 000 m/s (2 000 × g) for 15 min. The extract is preferably used immediately but may be stored for up to

48 h at a temperature of not more than 7 °C or frozen for up to 15 days at below −10 °C.

7.2.3 Procedure B — Glove-in-glove procedure

7.2.3.1 Take two gloves and determine the mass of each one to an accuracy of not less than 0,001 g (m

and m ). Mark each glove at a point on the cuff 20 cm from the tip of the middle finger. Take one glove and

insert it inside the other so that they fit together (this can be done conveniently using rods to insert the thumb

into the thumb, etc.; however, the method of doing this is not critical as long as the gloves are exposed to

minimum handling). Repeat the process with two further pairs of gloves of the same size.

7.2.3.2 Pour sufficient dye solution (6.1) into the inner glove to fill all of the fingers. Introduce 25 cm of

extractant (6.2) between the inner and outer glove. Manipulate gently to remove any air bubbles and seal the

gloves with a clamp (5.9) at the 20 cm mark.

7.2.3.3 Fix the gloves to a shaker and shake for 120 min ± 5 min at 25 °C ± 5 °C. If small droplets of

liquid are noted on the outer surface, suggesting the presence of pinholes in the outer glove, discard the

samples and repeat the extraction with a fresh pair of gloves.

7.2.3.4 Remove the clamp and separate the gloves carefully, taking care not to contaminate the extract

with the dye solution in the inner glove.

7.2.3.5 Decant the extract from the outer glove into a centrifuge tube (5.3). If it is coloured blue, it is

indicative of a pin-hole or cross-contamination. In such cases, discard the solution and repeat the extraction

with a fresh pair of gloves. Clarify the extract by centrifugation at not less than 20 000 m/s (2 000 × g) for

15 min. Store the extract at a temperature of not more than 7 °C and carry out the determination within 48 h.

Alternatively, frozen aliquots of the extract may be stored at −10 °C or lower for up to 15 days.

7.2.3.6 Cut both gloves at the 20 cm mark to remove the cuffs. Remove surplus liquid from the cuffs by

blotting and allow to dry at room temperature. Determine the mass of the cuffs (m ) to an accuracy of not less

than 0,001 g. Calculate the average mass (m ) of the extracted part of the gloves: m = (m + m − m )/2 where

s s 1 2 c

m and m are the masses of the original gloves and m is the combined mass of the un-extracted cuffs.

1 2 c
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ISO 12243:2003(E)
7.3 Preparation of standard protein solutions

Prepare standard solutions of protein by dilution of the protein stock solution (6.4) with extractant solution (6.2),

3 3 3 3 3

to make solutions with concentrations of e.g. 40 µg/cm , 20 µg/cm , 10 µg/cm , 5 µg/cm , and 2,5 µg/cm .

Use the extractant (6.2) as a blank. The solutions are stable for 2 days refrigerated (see the Note).

NOTE The lower concentrations can readily be prepared by two-fold serial dilution of the appropriate more

concentrated solution. The standard solutions should cover a wide range of concentrations the precise values of which are

known since the exact concentration of the stock solution has been determined (see 6.4). These solutions are also

required for the verification procedure described in Annex A.
7.4 Precipitation and concentration of protein
7.4.1 General
Carry out single determinations at 25 °C ± 5 °C.

7.4.2 Accurately transfer 4 cm each of extractant (6.2) (as a blank), the standard protein solutions

3 3

(see 7.3) and the three glove extracts to 10 cm centrifuge tubes (5.3). Add 0,4 cm of DOC (6.3.6), mix and

3 3

allow to stand for 10 min, then add 0,4 cm of TCA (6.3.7) and mix. Add 0,4 cm of PTA (6.3.8), mix (see

the Note) and allow to stand for a further 30 min.

NOTE The amount used is to ensure a sufficient quantity for analysis using a cuvette. If a micro-plate reader is used,

the quantities may be reduced proportionately. If a large number of samples is involved, it is particularly important to

ensure that the centrifuge tubes are clearly identified.

7.4.3 Centrifuge at not less than 60 000 m/s (6 000 × g) for 30 min. It is important that the protein is

properly compacted. If necessary, extend the time of centrifugation. Decant the supernatant liquid and drain

by inverting each centrifuge tube on an absorbent paper towel. Add 0,8 cm of 0,2 mol/l sodium hydroxide

solution (6.3.5) to each tube, including the blank, to redissolve the precipitated protein. Use a vortex mixer or

ultrasonic water bath (5.7) if needed.

Ensure that the protein has completely redissolved to give a clear solution. Should some protein precipitate

3 3

remain, add a further measured quantity of sodium hydroxide solution up to 3,2 cm (i.e. a total of 4,0 cm ).

The same amount of sodium hydroxide shall be used for each of the solutions. The recommended amount of

sodium hydroxide solution (0,8 cm ) gives a concentration factor F of 5. If the same amount of sodium

hydroxide is not used for each sample, then F will vary from one sample to another:

Volume of extract before precipitation
Volume of NaOH used to redissolve the protein

The redissolved-protein solution should preferably be used the same day. If the determination cannot be

carried out at once, the pellet may be stored for not more than 24 h at a temperature not exceeding 7 °C.

In cases where complete dissolution in not achieved after addition of 4,0 cm of NaOH, centrifuge at

60 000 m/s (6 000 × g) for 15 min to give a clear protein solution.
7.5 Colour development

7.5.1 Switch on the spectrophotometer and zero it in accordance with the manufacturer’s instructions.

3 3

7.5.2 To 0,8 cm of the redissolved-protein solutions, including the blank from 7.4.2, add 0,3 cm of

reagent A (6.3.1) and mix well. Add 0,1 cm of reagent B (6.3.2), mix, and allow to stand for at least 15 min

but no longer than 1 h before measuring the absorbance.

NOTE Only 0,8 cm of the redissolved-protein solution is used for the colour reaction, regardless of the final volume

of the redissolved-protein solution.
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ISO 12243:2003(E)

If precipitation occurs on standing due to the presence of certain interferants, centrifuge to give a clear

solution prior to colour measurement.
7.5.3 Spectrophotometric measurement

Transfer the solutions prepared in 7.5.2 to cuvettes and measure the absorbance versus the blank at 750 nm

(preferred) or a specific wavelength in the range 600 nm to 750 nm within 1 h of adding reagent B. For uniform

results, the time scales, equipment and chosen wavelength must remain consistent. Determine the protein

content, in micrograms per gram of glove, as described in 8.3.
7.5.4 Measurement using a micro-plate reader

Transfer 0,49 cm of the solutions prepared in 7.5.2 to a flat-bottom microtitre plate (see 5.10.2) and measure

the absorbance versus the blank at a specific wavelength in the range 600 nm to 750 nm within 1 h of adding

reagent B. Determine the protein content, in micrograms per gram of glove, as described in 8.3.

8 Calculation of results
8.1 Calibration curve

Prepare a calibration curve by plotting the concentration of the original protein solutions (see 7.3) against their

absorbance after undergoing precipitation and being redissolved (see 7.5.3 or 7.5.4).

NOTE Some protein is lost during the concentration process. The method assumes that the same percentage of

protein is lost from the standards as from the test samples during concentration.

8.2 Calculation of concentrations

Determine the concentration c of each of the three extracted samples, in micrograms per cubic centimetre of

extract, by using their absorbance to read them directly from the curve. Report the median value.

NOTE In the event that the calibration curve is non-linear, the value can be calculated by polynomial regression. It is

suggested that commercial computer software for curve fitting and calculation of unknown concentrations is more practical.

8.3 Calculation of extractable-protein content
8.3.1 Procedure A — Cut-glove procedure

Calculate the extractable-protein content E, in micrograms per gram of glove, from the equation:

Vc×× 5
F× m
where
V is the volume of extractant used, in cubic centimetres;

c is the protein concentration in the redissolved-protein solution, in micrograms per cubic centimetre;

F is the concentration factor;
m is the mass, in grams, of the whole glove.

NOTE The value of 5/F will be 1 unless it has been necessary to use other than the recommended amount of sodium

hydroxide — see comment in 7.4.3.
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ISO 12243:2003(E)

The extractable protein per glove, in micrograms, is obtained by multiplying the result obtained above by m:

Extractable protein per glove = E× m
8.3.2 Procedure B — Glove-in-glove procedure

Calculate the extractable-protein content E, in micrograms per gram of glove, from the equation:

Vc×× 5
F× m
where
V is the volume of extractant used, in cubic centimetres;

c is the protein concentration in the redissolved-protein solution, in micrograms per cubic centimetre;

F is the concentration factor;
m is the mass, in grams, of the glove sample extracted (see 7.2.3.6).

NOTE The value of 5/F will be 1 unless it has been necessary to use other than the recommended amount of sodium

hydroxide — see comment in 7.4.3.

The extractable protein per glove, in micrograms, is obtained by multiplying the result obtained above by m:

Extractable protein per glove = E× m
where
m is the mass, in grams, of a whole glove =+()mm/2;
 12 
m and m are the respective masses of the original pair of gloves.
1 2
8.3.3 Conversion to mass per unit surface area

Regulatory authorities may require the results to be expressed in terms of surface area, e.g. micrograms per

unit area. Conversion to these values is as follows:
Vc×× 5
Extractable protein in µg/dm =
F× A

where A is the total surface area of the glove (see 7.2.2.3), in square decimetres.

9 Precision
9.1 Background

An interlaboratory test programme (ITP) to evaluate the precision of the method was conducted in 2002 using

the precision procedures and guidelines described in ISO 9272 (in preparation). The existing ISO/TR 9272

may be consulted for other details and terminology.

Both extraction procedures were evaluated: the cut-glove procedure and the glove-in-glove procedure. The

ITP was conducted with four materials with increasing measurement levels. Seven laboratories participated in

the ITP, and a Type 1 precision was evaluated. A test result is the mean of three replicates on each of two

separate test days, and precision is given in terms of test results, i.e. a mean value for each of two test days.

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ISO 12243:2003(E)
The precision results as determined by this ITP shall not be applied to accep
...

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