Scottish Health Survey 2012 - Volume 2 Technical Report

Chapter 2: Quality control of Urine and saliva analytes

Marilyn Roth, Wissam Gharib, Alix Hampson, Julie Day, Mira Doig Shanna Dowling and Lisa Rutherford

2.4 Introduction and Key Conclusions

This section describes the assay of analytes for the 2012 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 described in Appendix B.

The overall conclusion for the data provided in this chapter is that methods and equipment used for the measurement of urine and saliva analytes produced internal quality control (IQC) and external quality assessment (EQA) results within expected limits. The results of the analyses for each of the main urine analytes and saliva cotinine levels were acceptable for the 2012 SHeS.

2.2 Analysing laboratory

As in previous years, the Royal Victoria Infirmary (RVI) in Newcastle upon Tyne was the analysing laboratory used in the 2012 SHeS for the urine sample analyses. Salivary cotinine analysis for 2012 was conducted by ABS Laboratories in Welwyn Garden City, Hertfordshire.

2.3 Samples collected

2.3.1 Urine samples

A mid-flow spot urine sample was obtained from adults (aged 16 and over), for analysis of sodium, potassium and creatinine. A special urine collection syringe was used for this purpose. Participants were given the option of drawing up the sample themselves using the syringe and an instruction card or allowing the interviewer to draw up the sample.

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.

2.4 Methodology

2.4.1 Laboratory procedures for urine samples

All analyses were carried out according to Standard Operating Procedures by State Registered Biomedical Scientists (BMS) under the supervision of the Senior BMS. All results were routinely checked by the duty Biochemist.

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. Records were kept of when maintenance was due and carried out.

2.4.2 Laboratory procedures for saliva samples

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.

2.4.3 Urine sample analytical methods and equipment

Urinary sodium, potassium and creatinine analysis was carried out in the Biochemistry Department at the RVI using a Roche Modular P analyser. Urinary sodium and potassium were analysed using the indirect ISE method. Urinary creatinine was analysed using the Jaffe method. A Roche Modular P analyser was used throughout the SHeS 2012. The Roche Modular P analyser has been used in SHeS since April 2010, prior to this an Olympus 640 analyser was used.

The effects of this change of equipment were that measured concentrations were on average lower by 1.0 mmol/L for urinary sodium, 4.0 mmol/L for urinary potassium and 0.8 mmol/L for urinary creatinine. The equipment change did not affect the potassium/ creatinine ratio results but sodium/creatinine ratio results were on average 1.0 mmol/mmol lower.

2.4.4 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 was a high performance liquid chromatography coupled to tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS).^[1] The sample preparation prior to LC-MS/MS was liquid/liquid extraction. A Tomtec Quadra was used to allow for the automation of some of the sample preparation. All methods were validated before use.

An advantage of the LC-MS/MS assay is that it is less prone than other methods to non-specific interference when assaying low levels of cotinine as seen due to passive smoking, and so is preferable for samples from non-smokers1.

A disadvantage of LC-MS/MS is that it does not have the dynamic range of the GC-NPD assay used in previous years1. Therefore in SHeS 2012 the laboratory was informed whether the samples were from self-reported smokers or not. All the samples from self-reported smokers were first assayed using the high calibration range assay of 1 to 1,000 ng/mL, and any that were below 1 ng/mL were then re-assayed with the low range assay. All the remaining samples were first assayed using the low range assay that quantified samples over the range 0.1 to 100 ng/mL. Any of these that were over-range were then re-assayed using the high calibration range assay of 1 to 1,000 ng/mL, provided there was sufficient saliva available from that participant.

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.^[2],[3],[4] Internal quality assessment results are available from ScotCen Social Research upon request.

2.5.3 Urine samples

Two levels of IQC were used for urinary sodium, potassium and creatinine. Quality control samples were run at the beginning of the day and at regular intervals throughout the day, as for the other parameters.

2.5.4 Saliva samples

ABS laboratories ran 16 non-zero calibration standards for each batch of the low range assay (0.1-100 ng/mL) or high range assays (1-1,000 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)

2.6.1 Introduction

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. The samples contain varying concentrations of analytes. The same samples may or may not be distributed more than once.

Samples are assayed shortly after they arrive at the laboratory. Depending on the frequency of distribution, there may be weeks or months in which no EQA samples are analysed. Results are returned to the scheme organisers, who issue a laboratory specific report giving at least the following data:

• Mean values, usually for all methods and for method groups;
• A measure of the between-laboratory precision;
• The bias of the results obtained by that laboratory.

EQA is a retrospective process of assessment of performance, particularly of inaccuracy or bias with respect to mean values; unlike IQC, it does not provide control of release of results at the time of analysis.

The United Kingdom National External Quality Assessment Schemes (UKNEQAS) is a network of EQA schemes run by UK clinical laboratories. The Welsh External Quality Assessment Schemes (WEQAS), the National External Quality Assessment Scheme for Haematology, and the Central Quality Assessment Schemes (QAS) are all schemes in which the laboratories participate on a routine basis.

Monthly EQA results are available upon request from ScotCen Social Research.

2.6.2 Urine samples

The Clinical Biochemistry laboratory participates in the WEQAS scheme for the urine analytes (sodium, potassium and creatinine).

2.6.3 Saliva samples

There was no external quality control scheme available in 2012 for cotinine analysis but ABS Laboratories participates in inter-laboratory split analyses to ensure comparable results. The latest International inter-laboratory study was published in 20091.

References and notes

¹ 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.

² 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 analyzed, which is common for applications in haematology. More detail is available at <www.westgard.com/mltirule.htm#westgard>

³ 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.

⁴ 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.