Lot40110

6,10

Fig. 21.4 Internal quality control. Results obtained measuring a certified control serum over one year by enzyme immuno assay (EIA). Shortly after introduction of this control serum it became evident that the assay had the tendency to overestimate the target value of this lot (17.9 nmol/l). After servicing of the pipettor/plate reader equipment and re-validation (August 2002) the results improved.

to the scheme organizer. The results are then evaluated and the laboratory receives an assessment of its performance. An example of the results of the external quality assessment survey in Germany, organized by the German Society of Clinical Chemistry is given in Fig. 21.5. This example and the data available from the UK National External Quality Assessment Scheme (UK NEQAS) and from the US College of American Pathologists (CAP) reveal that, overall, less than 15% of the labs still use RIA for testosterone measurement, direct non-extractive methods are employed almost exclusively. More than 60% of the labs perform the analysis using automatic multianalyzers. In the UK NEQAS 2002 and in the US CAP survey 2003 only 3/239 and 3/953 participants, respectively used an extraction/chromatographic method1. The data in Fig. 21.5 show the very high inter-lab variability of the results obtained in Germany from the measurement of two serum samples containing testosterone concentrations in the adult male range. Most of the laboratories manage to

1 College of American Pathologists' 2003 Ligand (Special) Survey Y-A. Participant Summary Report. All conclusions and interpretations in this publication with respect to the College of American Pathologists' database are those of the author and not those of the College.

RIA

EIA

20.3

17.1

20.3

17.1

12.2

Jg* •

12.2

f

4.07

7.32

24.5

57.3

4.07

7.32

24.5

57.3

13.5

40.9

68.2

13.5

40.9

68.2

FIA

LIA

20.3

17.1

20.3

17.1

12.2

12.2

4.07

7.32

24.5

57.3

4.07

7.32

24.5

57.3

Fig. 21.5 External quality assessment. Results of two control sera with a testosterone concentration of 12.2 nmol/l (sample A, Y axis) and 40.9 nmol/l (sample B, X axis), respectively as measured by mass spectrometry in German laboratories participating in an external quality assessment scheme. Results are grouped by radioimmunoassays (RIA), methods based on enzymatic (EIA), fluorimetric (FIA) or luminescent (LIA) detection systems. Each dot represents results from one laboratory. Target values are indicated by the small white square at the center of each graph. According to current German guidelines the target is considered to be successfully met if the results fall within ±40% of the value measured by mass spectrometry, indicated by the square defined by dotted lines. Results available online from the German Society of Clinical Chemistry (www.dgkc-online.de).

produce results falling within the allowed range, which, according to the actual German guidelines, permits variations of ± 40% of the testosterone value measured by mass spectrometry. This is, of course, a very wide range, such that the measurement of sample A of Fig. 21.5, which has a nominal value of 12.2 nmol/l, is considered successful if the laboratory obtains any value between 7.32 and 17.1 nmol/l.

In practice this means that a man with borderline serum testosterone concentrations has an equal probability of being classified as normal or hypogonadal (i.e. > or < 12 nmol/l) and both diagnoses are correct from the analytical point of view. The comparison between the four method groups shown in Fig. 21.5 does not reveal major differences among them. However, a more accurate analysis of external quality assessment results is usually performed by the UK NEQAS and this permits identification of kits which are less accurate than others (Middle 2002). The UK NEQAS analysis also shows clearly that the performance of testosterone kits is quite bad for the measurement of female samples, whereas the overall bias for male samples (about ± 15%) is acceptable. Therefore, as long as commercially available kits are used, the accuracy of the serum testosterone determination is very much dependent on the in-house re-validation and of a very strict internal quality control, both of which remains up to the individual laboratory.

External quality assessment for SHBG and free testosterone is much less advanced. UK NEQAS and US CAP offer schemes for SHBG, but only few laboratories make use of them. The US CAP survey 2003 reports on 79 laboratories measuring SHBG and 9 laboratories measuring bioavailable testosterone by ammonium sulfate precipitation. As far as free testosterone is concerned, six labs were reported to use equilibrium dialysis, three labs centrifugal ultrafiltration and 70 labs used an "analog" method. In view of the analytical problem of the "analog" kits reported above, it is not surprising that these labs produce free testosterone results much too low compared to those obtained by FT calculation, dialysis or ultrafiltration. No external quality assessment is presently available for DHT.

21.7 Key messages

• Serum testosterone is measured in clinical routine by immunological competitive methods based on polyclonal antisera and labeled hormone.

• The reference method for testosterone measurement is mass spectrometry. Extraction methods should be used for accurate, reproducible testosterone measurements.

• Non-extraction, direct methods based on non-radioactively labeled tracers are currently routinely used. Automatic multianalzers are those systems mostly used for serum testosterone measurement.

• Any testosterone measurement system, including those based on automatic analyzers, should be carefully validated in-house against an extraction method before it is adopted for routine assays.

• So-called "analog" free testosterone methods are unreliable. Calculated free testosterone gives the best estimation of free testosterone as measured by dialysis or ultrafiltration.

• DHT should be measured by chromatographic or oxidative methods.

• Participation in external quality control programs is mandatory. Strict internal quality control is fundamental to ensure accurate measurements.

21.8 REFERENCES

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