MONICA Manual, Part III: Population Survey

Section 3: Standardization of thiocyanate measurements

November 1990

This section provides details on the methods to be used for standardization of thiocyanate measurements in the WHO MONICA Project.

Contents

• Introduction
• 1. Determination of thiocyanate in serum
  • 1.1 Automated method for autoanalyzer II
  • 1.2 Manual method
  • 1.3 SCN laboratory performance guidelines
• References

• Copyright notice
• Manual acknowledgements
• Document identification:
  • URL:http://www.ktl.fi/publications/monica/manual/part3/iii-3.htm
  • URN:NBN:fi-fe19981153

Queries/comments on this section should be addressed to:

Introduction

Smoking habits, e.g. inhalation, may vary locally and with time; all may not be reflected in the information collected by the smoking questionnaire. Moreover, socio-cultural factors may affect the truthfulness of the answers. Among the objective variables for validating smoking questionnaire responses, carboxyhaemoglobin has a half-life of 3-4 hours and serum thiocyanate of 10-14 days. Thiocyanate is thus less subject to short-term variation in smoking. Moreover, serum thiocyanate has been validated as a predictor of total and coronary 15-year mortality, being either superior or equal to the smoking questionnaire in two populations, respectively [1].

Among non-smokers the standard deviation of thiocyanate was found to be approximately 25% of the mean, and 15% among those smoking 10 or more cigarettes  a day. The "baseline" level of thiocyanate is also affected by the diet.

In MONICA, thiocyanate determinations may serve two purposes:

1. to validate the smoking questionnaire data for the population and its subgroups, and
2. to provide an aggregate measure of trends in population exposure to smoking.

For (a), a sufficient number of data for each sex, age and smoking category should be collected, while for (b) four groups only at the initial and the final surveys, respectively, would be the minimum requirement: male versus female, non-smokers versus present smokers. The non-smokers are necessary for showing trends in the background level dependent on the diet. Only the complete coverage of a MONICA survey sample would provide sufficient accuracy for (a), while for (b) as little as a 20% random sample would be suffice. For the validation of population trends in smoking, thiocyanate obviously should be analyzed at least twice, both at the initial and the final surveys. On the other hand, for a stratified study of the validity of the questionnaire - or comparing questionnaires- the mid-point survey would have some merit in providing data for extrapolation over the 10-year period. MCCs are encouraged to measure thiocyanate on all subjects even if this was not possible in the initial survey.

1. Determination of thiocyanate in serum

When ferric ions in acid solutions are added to thiocyanate, a brownish-red complex of ferric cyanate with an absorption peak at 455 nm is formed. Based on this reaction, RG Bowler published a method to determine thiocyanate in serum [2]. Later, this method was adapted for Autoanalyzer II by William C. Butts et al [3]. Both are given here with the following modifications:

1. In the automated method of Butts:
   • the determination is run at 60 samples per hour with a sample-to-wash ratio of 8:2.
   • better absorbances are obtained when the usual flow-cell of 15 mm light-path is replaced by a 50 mm cell.
2. In the manual method of Bowler, the following alterations are suggested:
   in order to obtain higher absorbances, the amount of sample is increased to 2 ml to give a final dilution of 1:3 in the reaction mixture, and
   • the volumes of diluent and TCA (trichloroacetic acid) are decreased,
   • the final ferric nitrate concentration is maintained at less than 10 g/l, as at higher concentrations the absorption peek at 455 nm is distorted.

1.1 Automated method for autoanalyzer II

Reagents:

Ferric nitrate, 10 g/l. Dissolve 10 g of Fe (NO₃ )₃·9 H₂O p.a. in 1.5-M nitric acid and dilute to one litre with the same acid. Filter, store at room temperature in a dark bottle. Prepare every two weeks.

Sodium perchlorate, 0.1-M. Dissolve 12.25 g of NaClO₄ p.a. in de-ionized water and dilute to one litre with water. Filter, add 1 ml of Brij-35 (300 g/l), mix, store in a dark bottle at +4°C.

Dialyzer recipient. To one litre of de-ionized water add 1 ml of Brij-35 (300 g/l), mix well.

 

Standards:

Stock thiocyanate standard, 1000 µmol/l. Dissolve 97.2 mg of potassium thiocyanate (KSCN, p.a.) in de-ionized water and make up the solution to one litre with water. The standard is stable at room temperature for two to three months.

Working standards of 50, 100, 150, 200 and 250 µmol/l are prepared from the stock standard by diluting with water. Working standards are not stable for more than two weeks at room temperature. Secondary standards, prepared by adding known amounts of thiocyanate to a serum pool can also be used instead of primary water standards if the apparent thiocyanate concentration of the so-prepared pool had been determined reliably on the basis of primary standards (remember that the serum pool would show its own "thiocyanate" concentration which will be summed with the added thiocyanate concentration).

 

Controls:

It is recommended that a serum pool from the blood of smokers be prepared and the control serum in suitable portions. A frozen control stored at -20°C can be used for at least six months.

1.2 Manual method

Reagents:

Ferric nitrate, 20 g/l. Dissolve 20 g of Fe (NO₃ )₃·9 H₂O p.a. in 1.5-M nitric acid and dilute to one litre with the same acid. Filter, store at room temperature in a dark bottle. Prepare every two weeks.

Sodium perchlorate, 0.1-M. Dissolve 12.25 g of NaClO₄ p.a. in de-ionized water and dilute to one litre with water. Filter, and store in a dark bottle at +4°C.

Trichloroacetic acid, 20 per cent. Dissolve 20 g of TCA in 100 ml of de-ionized water, filter. Store at room temperature.

 

Standards:

Stock thiocyanate standard, 1000 µmol/l. Dissolve 97.2 mg of potassium thiocyanate (KSCN, p.a.) in de-ionized water and make up the solution to one litre with water. The standard is stable for two to three months at room temperature.

Working standards of 50, 100, 150, 200 and 250 µmol/l are prepared from the stock standard by diluting with water. Working standards are not stable for more than two weeks at room temperature.

Working scheme:

Into a centrifuge tube (preferably conical) pipette:

Mix vigorously, leave to stand for 20 minutes at room temperature, then centrifuge down the precipitated protein. The supernatant must be clear (non-turbid). Centrifugation at 5000 G or more may be necessary.

Take:
clear supernatant	2 ml
Ferric nitrate 20g/l solution	1 ml

Mix and measure the absorbance at 455 nm (using a spectrophotometer) or at a wavelength in close vicinity (using a filter photometer) immediately, or within a few minutes after mixing (if a series of samples is measured). The colour begins to fade after 30 minutes in sunlight (which occurs more rapidly in standards than in samples).

1.3 SCN laboratory performance guidelines

Due to the broad variability of measurement procedures and the purpose to which the SCN data is put, the guidelines for laboratory performance, relating to quality control sample reference values, are less stringent than those for cholesterol. Bias should not exceed 10% of the reference values. The CV(%), as measured by the standard deviation of the individual laboratories determination for a given pool divided by the reference value for that pool and multiplied by 100, should be no greater than 5 for any pool.

References

1. Heliövaara, M. et al. Serum thiocyanate concentration and cigarette smoking in relation to overall mortality and to deaths from coronary heart disease and lung cancer. J Chron Dis 1981;34:305-11.
2. Bowler RG.  Biochem 1944;38:385-388.
3. Butts WC et al. Clin Chem 1974;20:1344-1348.