The value of microscopy analysis

Garry BurdettHealth and Safety Laboratory

Harpur Hill, Buxton, UK, SK17 9JN

Asbestos Hazard

• Mortality (excess deaths) due to:– Fibrosis of the lung (asbestosis)– Cancer of the lung (bronchial cancer etc)– Cancer of the lung lining (e.g. mesothelioma)

• Morbidity (lung changes/dysfunction)– Pleural plaques;– Diffuse pleural effusions

Fibre Related Risk:

• Depends on:– Dimensions– Durability / Type– Dose / Exposure– Time

(Measurable by microscopy)

EU Directive: Dimensions

• For the purposes of measuring in the air, only fibres with a length of more than five micrometres, a breadth of less than three micrometres and a length to breadth ratio greater than 3:1 shall be taken into consideration.

(2003/18/EC amending to EU asbestos worker protection directive (83/477/EEC) article 7.6 )

Durability / Type

500

100

10

100

200

300

400

500

Ris

k fa

ctor

Crocidolite Amosite Chrysotile

Asbestos type

Fig 1: Mesothelioma risk by fibre type

Dose / Exposure

• Exposure is used as a surrogate for dose by sampling a known volume of air onto a membrane filter and counting the numbers of fibres present in a known area.

• This is used to calculate the fibre concentration in f/ml or f/cm3 of air.

• Uses size criteria to determine ability to reach pulmonary region of the lung.

Time

• Dose = exposure x duration (f/ml/years)• Risk cumulative dose (assume linear);• Risk time since first exposure

(mesothelioma related by power law).• Lag time 15 – 60 years from exposure to

disease symptoms

EU directive: Fibre Counting

• “Fibre counting shall be carried out whenever possible by PCM (Phase contrast microscope) in accordance with the 1997 (World Health Organisation) recommended method (16) or any other method giving equivalent results.”

EU Directive: Requirements

• The 2003 amendment to 83/477/EEC abandoned action levels and different control levels for different types of asbestos and introduced a single limit of 0.1 f/ml over an 8-hour time weighted average. (Article 8)

• Therefore the EU regulation for controlling the risk to workers, require only PCM fibre number information over a broadly defined size range.

• Article 4 requires the type and quantities of asbestos handled to be notified

Main types of microscopy for fibre counting.

• Phase contrast light microscopy PCM • Scanning electron microscopy SEM• Transmission electron microscopy TEM.• All have ISO or WHO standard methods

• Polarised light microscopy PLM for bulk analysis.

PCM (X500 magnification)

SEM

TEM

SEM higher Mag & 3D

TEM high mag chrystotile

PLM : Dispersion staining

Discrimination of fibre type

• PCM: No discrimination or identification ;• PLM Uses optical properties + refractive

index/ dispersion colours to identify fibres; >0.8µm width;

• SEM uses Energy Dispersive X-ray analysis to semi-quantitatively measure chemistry;

• TEM uses quantitative EDX and electron diffraction to identify fibres.

Comparison of methods for fibre counting

Operating Mag

Area (mm2) examined

Time taken(mins.)

Rate area examinedmm2/hr

PCM X500 (200 fov) 1.5

20 4.5

SEM X2000 (50 fov)0.15

30 0.3

TEM x5000 (50 go)0.5

40 0.75

Likely upper limit of detection (f/ml) by method and volume

WORK ENVIRONMENTAL

COST PER HOUR

VOLUME(litres)

500 3000

PCM 0.01* 0.0017* 30-50

SEM 0.015 0.0025 80 - 120

TEM 0.005 0.0007 80 - 120

Accuracy & Precision

• Accuracy not usually known.

• Precision includes:– Random “Poisson” counting error– Instrumental errors (QA / calibration)– Human errors (QA / Proficiency testing)

Random counting errors (95%)

No. of fibres

Lower limit

Upper limit

No. of fibres

Lower limit

Upper limit

0 0 2.99 5 1.624 11.669

1 0.025 5.572 6 2.202 13.060

2 0.242 7.225 10 4.795 18.39

3 0.619 8.767 20 12.217 30.889

4 1.090 10.242 50 37.112 65.919

Quality and accreditation

• Quality assurance and proficiency testing is essential, particularly if results from different analysts and laboratories are being used or compared.

• Accreditation of Laboratories to ISO 17025 mandated by the EU directive.

Type of analysis

PCM SEM TEM PLM

Fibre counting of air samples 

Belgium (25)France (?)Netherlands(20)Spain (20)UK (200) 

Germany*  

France (20) Not applicable

Fibre Identification of bulk samples

Not applicable UK (5) UK (17) UK (250)

Fibre counting and identification of air samples

Not applicable Germany* 

France (20) Not applicable

* PT scheme run for one or two rounds in past a new scheme was plannedNote: UK schemes also include a number of EU countries who participate

RICE: Fibre counting PT

• RICE - 187 labs.• MMMF – 12 labs.• ERM – WHO major change

Chart 1. RICE Ratings

196 197 195 192 190 193 194 186 189 190 184 184 183 184 184

0

50

100

150

200

250

52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Round

Num

bers

of l

abor

ator

ies

Rating 1

Rating 2

Rating 3

AIMS: Fibre Identification PT

Chart 4 : Distribution of cumulative scores for rounds 22 - 24 (UK Laboratories)

25%

33%

19%

22%

1%

0 (No Errors)

7 (1 Minor Error)

8 - 20

21 - 47

> 47

Value of PCM counting

1. Workplace exposure can be controlled using the WHO - PCM method;

2. Counts “regulated” WHO fibres & meets EU criteria;

3. Relatively quick (on-site) and cheap;4. Environmental exposure can be

screened by PCM.

PCM Limitations

1. PCM has limited detection due to background counts on blank filters (0.01 f/ml);

2. No knowledge of fibre types;3. Only an “index of exposure”

Value of analytical SEM

• Can be adjusted to give regulated fibre counts,

• Can give fibre size information,• Classifies fibre types present using EDX

spectra data.

SEM limitations

• Limited availability;• Off site analysis;• Costly • Can only be used for regulated fibre

counting,• No definitive identification of fibres,• Lowest area of filter scanned per field of

view.

Value of TEM

• Can count regulated fibres + all fibre sizes.• Accurate size information,• EDX chemical analysis of all fibre sizes • Crystal structure information (SAED)• Identification of fibre type,• Can give complete information on all fibre

sizes if needed

Limitations of TEM

• Limited availability;• Off site;• Costly

Recommendations 1

• Use PCM for workplace control and checking after clean –up (with disturbance sampling).

• Indoor and near source environmental use PCM screening and SEM or TEM analysis (as necessary) on selected half-filters.

• Ambient air: Analytical TEM is best, SEM is more limited, PCM not very useful.

Recommendations 2

• Use Polarised light microscopy (PLM) for analysis of bulk samples for asbestos present and type of asbestos.

• Use PLM to screen minerals and soils for asbestos content.

• Asbestos in water usually analysed by TEM