The Scottish Health Survey 2019- volume 2: technical report

Presents information on the methodology and fieldwork from the Scottish Health Survey 2019.

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Chapter 2: Quality Control of Saliva Analytes

Julie Day, Mira Doig & Vicky Wilson

2.1 Introduction and Key Conclusions

This section describes the assay of analytes for the 2019 Scottish Health Survey (SHeS) biological samples and the quality control and quality assessment procedures that were carried out during the survey period. Details of procedures used in the collection, processing and transportation of the specimens are available on request from ScotCen Social Research.

The overall conclusion for the data provided in this chapter is that methods and equipment used for the measurement of saliva analytes produced internal quality control (IQC) and external quality assessment (EQA) results within expected limits. The results of the analyses for the saliva cotinine levels were acceptable for SHeS 2019.

2.2 Analysing Laboratories

As in previous years, the saliva samples were initially sent to the Royal Victoria Infirmary (RVI) in Newcastle upon Tyne where they were checked for correct identification and stored prior to dispatch to ABS Laboratories in Welwyn Garden City, Hertfordshire. ABS Laboratories conducted the analysis of salivary cotinine.

2.3 Samples Collected

2.3.1 Urine samples

In previous years a mid-flow spot urine sample was obtained from participants. Following the Scottish Government’s consultation on the questionnaire content of SHeS[1], it was decided that urine sampling should be removed from the survey from 2018. As a result, no urine samples were collected in 2019.

See the Scottish Health Survey 2017: Volume 2 Technical Report[2] for further details on urine sampling.

2.3.2 Saliva samples

A saliva sample was obtained from participants aged 16 and over. Saliva samples were collected for analysis of cotinine (a metabolite of nicotine that shows recent exposure to tobacco smoke). A saliva collection tube was used for this purpose. Participants were also offered the option to provide the saliva sample using a dental roll that they could saturate with their saliva before it was placed in the tube. The saliva tube was then labelled and packaged ready for dispatch.

2.4 Methodology

2.4.1 Laboratory procedures for saliva sample analyses

All analyses were carried out according to Standard Operating Procedures by analysts in a MHRA Good Laboratory and Good Clinical Practice (GLP & GCP) accredited laboratory. All work is reviewed by the Laboratory & QA Manager.

A schedule of Planned Preventative Maintenance was used for each item of analytical equipment. These plans were carried out jointly by the manufacturers and the laboratories’ staff. Records were kept of when maintenance was due and carried out.

2.4.2 Saliva sample analytical methods and equipment

Saliva samples received at the RVI were checked for correct identification, assigned a laboratory accession number, and stored at 4°C. Samples were checked for details and despatched fortnightly in polythene bags (20 samples per bag) by courier for overnight delivery to ABS Laboratories, where cotinine analysis was carried out. This laboratory specialises in accurate measurement of low levels of cotinine and therefore takes special precautions to ensure no contamination by environmental tobacco smoke occurs.

The method of analysis used since the 2009 SHeS study is high performance liquid chromatography coupled to tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS), replacing the gas chromatography nitrogen phosphorous detection (GC-NPD) method used in earlier SHeS studies[3]. The sample preparation prior to LC-MS/MS was liquid/liquid extraction. Samples were divided for analysis into batches of self-reported smokers and non-smokers and analysed either using a method with a high calibration range, 1 to 750 ng/mL for the self-reported smokers, or low calibration range 0.1 to 50 ng/mL for the non-smokers. A Tomtec Quadra was used to allow for the automation of some of the sample preparation. All methods were validated before use. If any of the samples from self-reported smokers gave a result below 1 ng/mL on initial analysis they were repeated in a low range batch. Similarly, if any of the non-smoker samples gave a result above 50 ng/mL then they were repeated in a high range batch.

2.5 Internal Quality Control (IQC)

2.5.1 Explanation of IQC

The purpose of internal quality control (IQC) is to ensure reliability of an analytical run. IQC also helps to identify, and prevent the release of, any errors in an analytical run. IQC is also used to monitor trends over time.

For each analyte or group of analytes, the laboratory obtains a supply of quality control materials, usually at more than one concentration of analyte. Target (mean) values and target standard deviations (SD) are assigned for each analyte. Target assignment includes evaluation of values obtained by the laboratory from replicate measurements (over several runs) in conjunction with target values provided by manufacturers of IQC materials, if available. The standard deviation and the coefficient of variation (CV) are measures of imprecision and are presented in the tables. IQC values are assessed against an acceptable range and samples are re-analysed if any of the Westgard rules have been violated[4],[5] [6]. Internal quality assessment results are available from ScotCen Social Research upon request.

2.5.2 Saliva samples

ABS laboratories ran 16 non-zero calibration standards for each batch of the low range assay (0.1-50 ng/mL) or high range assays (1-750 ng/mL). Six quality control (QC) samples, two each at a set concentration to represent low, medium and high levels for the calibration range being used, were also analysed with each analytical batch. For the results from any analytical batch to be acceptable, four out of the six QCs must have a bias of no greater than ±15% with at least one from each QC level being within these acceptance criteria, and 75% of the calibration standards must have a bias of no greater than ±15% except at the lower limit of quantification where the bias must be no greater than ±20%.

2.6 External Quality Assessment (EQA)

External quality assessment (EQA) permits comparison of results between laboratories measuring the same analyte. An EQA scheme for an analyte or group of analytes distributes aliquots of the same samples to participating laboratories, which are blind to the concentration of the analytes. The usual practice is to participate in a scheme for a full year during which samples are distributed at regular frequency (monthly or bimonthly for example); the number of samples in each distribution and the frequency differ between schemes.

There was no external quality control scheme available in 2019 for saliva cotinine analysis but ABS Laboratories participates in inter-laboratory split analyses to ensure comparable results. The latest International inter-laboratory study was published in 2009[3].

References and notes

1 Questionnaire Content of the Scottish Health Survey (2017): Consultation Analysis Report. Available from

2 Scottish Health Survey 2017: Volume 2 Technical Report. Available from

3 Bernert JT, Jacob III P, Holiday DB et al. Interlaboratory comparability of serum cotinine measurements at smoker and nonsmoker concentration levels: A round robin study. Nicotine Tob Res. 2009;11:1458-66.

4 Westgard rules are a statistical approach to evaluation of day-to-day analytical performance. The Westgard multirule quality control procedure uses five different control rules to judge the acceptability of an analytical run (rather than the single criterion or single set of control limits used by single-rule quality control systems, such as a Levey-Jennings chart with control limits set as either the mean plus or minus 2 standard deviations or the mean plus or minus 3 standard deviations). Westgard rules are generally used with two or four control measurements per run. This means they are appropriate when two different control materials are measured once or twice per material, which is the case in many chemistry applications. Some alternative control rules are more suitable when three control materials are analysed, which is common for applications in haematology. More detail is available at

5 Westgard JO, Barry PL, Hunt MR, Groth T. A multi-rule Shewhart chart for quality control in clinical chemistry. Clin Chem. 1981;27:493-501.

6 Westgard JO, Klee GG. Quality Management. Chapter 16 in Burtis C (ed.). Fundamentals of Clinical Chemistry. 4th edition. Philadelphia: WB Saunders Company, 1996, pp.211-23.



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