The Scottish Health Survey 2011 - volume 3: technical report

Chapter 2: Quality control of blood, urine and saliva analytes

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

2.1 INTRODUCTION AND KEY CONCLUSIONS

This section describes the assay of analytes for the 2011 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 blood, 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 blood and urine analytes and saliva cotinine levels were acceptable for the 2011 SHeS.

The analytical equipment used to measure C-reactive protein in blood samples changed during the survey period. This is described below.

2.2 Analysing laboratory

As in previous years, the Royal Victoria Infirmary (RVI) in Newcastle upon Tyne was the analysing laboratory used in the 2011 SHeS for the blood and urine sample analyses. Salivary cotinine analysis for the 2011 SHeS was conducted by ABS Laboratories in Welwyn Garden City, Hertfordshire.

2.3 Samples collected

2.1.3 Non-fasting blood samples

Following written consent from eligible participants, three non-fasting blood samples were collected for adults 16 and over (one 6 ml plain, a 4 ml EDTA and a 4.5 ml citrate tube). Children were not eligible to take part in nurse interview so no blood samples were collected for those aged under 16. The order of priority for collecting samples was firstly into the 6 ml plain tube (no anticoagulant) followed by the 4 ml EDTA (ethylene diamine tetra-acetic acid) tube, followed by the 4.5 ml citrate tube. After collection the tubes were despatched to the Department of Clinical Biochemistry at RVI, which acted as the co-ordinating department for transport of samples to the individual departments undertaking the analysis.

Samples collected in the 6 ml plain tube for serum

This provided the sample for total cholesterol, high density lipoprotein (HDL)-cholesterol, C-reactive protein and vitamin D analysis. If written consent was given by the participant, a minimum of 0.5 ml of the remaining serum was stored in a freezer at -40°C (±5°C) for possible future analysis.

Samples collected in the 4 ml EDTA (ethylene diamine tetra-acetic acid) tube

This provided the sample for the glycated haemoglobin analysis. If written consent was given by the participant, aliquots containing approximately 1ml of whole EDTA blood were processed for storage (unseparated) in a freezer at -20°C (± 5°C) for possible future analysis.

Samples collected in the 4.5 ml citrate tube

Samples in the citrate tube were used for fibrinogen analysis.

2.1.4 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.

2.1.5 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.

2.2 METHODOLOGY

2.2.1 Laboratory procedures

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 and highly abnormal results were notified to the Survey Doctor. The Survey Doctor notified and advised the participant and, where prior consent had been obtained, their general practitioner as appropriate.

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.

Figure 2A shows reference ranges used for each of the blood analytes measured in the 2011 SHeS. Values within these reference ranges were considered to be clinically 'normal' while those outside were treated as clinically 'abnormal' (either too high or too low). For total and HDL-cholesterol, where a large proportion of the population have values which are statistically within the normal distribution but are not ideal for good health, the term 'desirable' rather than 'normal' was used when results were sent to participants and/or their GPs.

Figure 2A

Figure 2A: Reference intervals for blood analytes^a

Analyte	Reference intervalb	Units
Serum Total cholesterol
Males	3.5-5.1	mmol/L
Females	3.5-5.1	mmol/L
HDL-cholesterol
Males	0.9-1.4	mmol/L
Females	1.1-1.7	mmol/L
Vitamin Dc
Males	See footnote b	nmol/L
Females	See footnote b	nmol/L
Blood Total glycated haemoglobin (HbA1c)d
Males	Non diabetic, <6.1	%
Females	Non diabetic, <6.1	%
Males	Non diabetic, <43	mmol/molHb
Females	Non diabetic, <43	mmol/molHb
C-reactive proteind
Males/Females	<0.5	mg/L
Fibrinogen
Males/Females	2.1 - 4.8	gl

^a Biochemistry and haematology laboratories, Royal Victoria Infirmary, Newcastle-upon-Tyne.
^b Vitamin D deficiency <25nmol/L. Osteomalacia likely at levels <15 nmol/L. Vitamin D levels of 25-50 nmol/L may indicate insufficiency and should be interpreted in conjunction with PTH and calcium levels. Serum levels vary with exposure to sunlight, peaking in the summer months.
^c From 2010, glycated haemoglobin results are presented as % and mmol/molHb values.
^d Reference range for high sensitivity CRP assay.

2.2.2 Blood sample analytical methods and equipment

The analytical equipment used to measure C-reactive protein in blood samples changed during the survey period as indicated in the sections below.

Total cholesterol

Measurement of total cholesterol was carried out in the Biochemistry Department at the RVI using a Cholesterol Oxidase assay method. A Roche Modular P analyser calibrated to the Centre for Disease Control (CDC) guidelines was used throughout SHeS 2011. The Roche Modular P analyser has been used in SHeS since April 2010, prior to this an Olympus 640 analyser was used.

The effect of this change of equipment was that measured concentrations of total cholesterol were on average 0.1mmol/L higher.

HDL-cholesterol

HDL-cholesterol analysis was carried out in the Biochemistry Department at the RVI using a direct method (no precipitation). A Roche Modular P analyser was used throughout SHeS 2011. The Roche Modular P analyser has been used in SHeS since April 2010, prior to this an Olympus 640 analyser was used.

The effect of this change of equipment was that measured concentrations of HDL-cholesterol were on average 0.1mmol/L lower.

C-reactive Protein (CRP)

Measurement of CRP was carried out by the Biochemistry Department at RVI using a Latex enhanced mono immunoassay. Initially a Behring Nephelometer II was used and from May 2011 the Roche Modular P analyser was used. The effect of this change of equipment was that measured concentrations of CRP were on average 0.15mmol/L higher.

Glycated haemoglobin

Glycated haemoglobin (HbA1c) analysis was carried out in the Biochemistry Department at the RVI using the Tosoh G8 analyser throughout SHeS 2011. The Tosoh G8 analyser has been used in SHES since 26^th August 2010; prior to this a Tosoh G7 analyser was used. There was no impact on measured concentrations. Both were calibrated using Diabetes Control and Complications Trial (DCCT) standards until October 2011 after which the Tosoh G8 was calibrated using the new recommended calibrator specific for HbA1c prepared by the International Federation of Clinical Chemistry (IFCC). DCCT aligned values were calculated from IFCC values.

Fibrinogen

Fibrinogen analysis was carried out in the Department of Haematology at RVI using the Auto Coagulation lab (TOP) CTS analyser. The modification of the Clauss thrombin clotting method was used.

Vitamin D

Serum 25-OH Vitamin D was measured using the Diasorin Liaison chemiluminescent immunoassay method.

2.2.3 Urine sample analytical methods and equipment

Sodium, potassium, creatinine

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 2011. 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.

2.2.4 Saliva sample analytical methods and equipment

Cotinine

Saliva samples received at the RVI were checked for correct identification, assigned a laboratory accession number, and stored at 40C. 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-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 2011 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 10 to 1,000 ng/mL, and any that were below 10 ng/mL were then re-assayed with the low range assay. In October 2011 the calibration range of the high range assay was extended to 1 to 1,000 ng/mL so that any samples from self-reported smokers that were below 1 ng/mL were re-assayed with the low range assay. All the remaining samples were first assayed using the low range assay of 0.1 to 100 ng/mL. Any of these that were over-range were then re-assayed using the high calibration range assay of 10 to 1,000 ng/mL (1 to 1,000 ng/mL from October 2011), provided there was sufficient saliva available from that participant.

2.3 INTERNAL QUALITY CONTROL (IQC)

2.3.1 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 upon request from ScotCen Social Research.

2.3.2 Non-fasting blood samples

Total and HDL-cholesterol

Low, medium and high control materials were assayed throughout the day.

C-reactive protein

Based on materials in use in the department, the Biochemistry department at RVI aim to achieve levels of reproducibility comparable to company literature, i.e. a coefficient of variation (CV) below 3%. However, realistically the imprecision at the low end of the analytical range leads to a CV of about 6%. Four levels of IQC are run at the beginning and end of each batch of samples on the Behring Nephelometer II Analyser or two levels were assayed at regular intervals throughout the day on the Roche Modular P analyser.

Glycated haemoglobin (HbA1c)

The analytical methods used for glycated haemoglobin measurement in the United Kingdom are now recommended to be standardised to a new standard specific for HbA1c prepared by the International Federation of Clinical Chemistry (IFCC). From October 2011 the IQC results for glycated haemoglobin are reported in IFCC standardised units of mmol/mol, before this date DCCT (National Glycohaemoglobin Standardisation Program) aligned values (%) were reported. Two levels of internal quality control were run at the beginning and end of each run and at regular intervals throughout.

Fibrinogen

Control plasmas are assayed at regular intervals and instrument function tests are monitored continuously for fibrinogen with the control interval specified as every 12 hours. Significant deviations from specified limits are flagged and must be acknowledged by the operator.

Vitamin D

Two levels of internal quality control were run at beginning and end of each run and then regular intervals throughout.

2.3.3 Urine sample

Sodium, potassium and creatinine

Two levels of IQC were used for urine 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.

2.3.4 Saliva samples

Cotinine

ABS laboratories ran 16 non-zero calibration standards for each batch of the low range assay (0.1-100 ng/mL), and 12 for the high range assay (10-1,000 ng/mL). During 2011 the high ranges assay was modified and a new calibration range set up and validated. In October 2011 the high range assay calibration range was extended to 1-1,000 ng/mL using 16 non zero calibration standards and the low QC were lowered to 3 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%.

2.4 EXTERNAL QUALITY ASSESSMENT (EQA)

2.4.1 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. DEQAS is an EQA scheme for Vitamin D.

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

2.4.2 Non-fasting blood samples

Total cholesterol

The Clinical Biochemistry laboratory participates in the WEQAS schemes.

HDL-cholesterol

The Clinical Biochemistry laboratory participates in the WEQAS scheme.

Glycated haemoglobin

The Clinical Biochemistry laboratory participates in the WEQAS scheme.

Fibrinogen

The Haematology laboratory participates in Central QAS schemes fortnightly and the NEQAS scheme quarterly.

C-reactive Protein

The Clinical Biochemistry laboratory participates in the 'WEQAS high sensitivity CRP scheme'.

Vitamin D

The Clinical Biochemistry laboratory at the RVI participates in the DEQAS scheme.

2.4.3 Urine samples

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

2.4.4 Saliva samples

Cotinine

There was no external quality control scheme available in 2011 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