Scottish Health Survey 2013 - 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, Tracey Maw and Lisa Rutherford

2.1 INTRODUCTION AND KEY CONCLUSIONS

This section describes the assay of analytes for the 2013 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 2013 SHeS.

2.2 ANALYSING LABORATORIES

As in previous years, the Royal Victoria Infirmary (RVI) in Newcastle upon Tyne was the analysing laboratory for the urine sample analyses in 2013. Salivary cotinine analysis of the 2013 samples 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 participants aged 16 and over taking part in the biological module. Urine samples were collected for analysis of sodium, potassium and creatinine. Participants were instructed to provide a sample of urine in the disposable collection beaker and then use the special urine collection syringe to draw up the sample. An instruction card was given to participants demonstrating how to use the syringe. Interviewers could also draw up the sample from the beaker if the participant preferred this. The urine collection syringe was then labelled and packaged ready for dispatch.

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 urine sample analyses

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 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.

2.4.3 Urine sample analytical methods and equipment

Urinary sodium, potassium and creatinine analysis was carried out in the Blood Sciences (formerly 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 2013. The Roche Modular P analyser has been used in SHeS since April 2010, prior to this an Olympus 640 analyser was used.

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). 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-smokers^[1].

A disadvantage of LC-MS/MS is that it does not have the dynamic range of the GC-NPD assay used in previous years.^[1] Therefore in SHeS 2013 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.

In November 2013 the calibration range of the high range assay was changed to 1-750 ng/mL as it was a more appropriate range for the cotinine levels seen in the majority of the samples and better linearity was achieved on the calibration curve over this reduced range. As a consequence the medium QC concentration was lowered to 200 ng/mL and the high QC to 500 ng/mL.

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.

2.5.2 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.

2.5.3 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 Welsh External Quality Assessment Schemes (WEQAS) are 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 Blood Sciences (formerly 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 2013 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 2009.^[1]