CHAPTER 2: QUALITY CONTROL OF BLOOD, URINE AND SALIVA ANALYTES
Catherine Bromley and Martine Miller
2.1 INTRODUCTION AND KEY CONCLUSIONS
This section describes the assay of analytes, quality control and quality assessment 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 about the data provided in this chapter is that the methods and equipment used for the measurement of blood, saliva, and urine 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 2008 SHeS.
2.2 ANALYSING LABORATORY
As in 1998 and 2003, the Royal Victoria Infirmary ( RVI) in Newcastle upon Tyne was the main analysing laboratory used in the 2008 Scottish Health Survey for the blood and urine samples analysis. Cotinine analysis for the 2008 Scottish Health Survey was conducted by ABS Laboratories, London, as it has been in previous years.
2.3 SAMPLES COLLECTED
2.3.1 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 visits 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 tube and the 4.5 ml citrate tube. After collection the tubes were despatched to the Department of Clinical Biochemistry at RVI who 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 lipid ( HDL)-cholesterol, and C-reactive protein analysis. If written consent was given by the informant, a minimum of 0.5 ml of the remaining serum was stored in a freezer at -70°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 informant, approximately 1 ml of whole blood was stored in a freezer at -20°C (±5°C) for possible future analysis.
Samples collected in the 4.5 ml citrate tube
This provided the sample for fibrinogen analysis.
A mid-flow spot urine sample was obtained from participants aged 16 and over, for analysis of sodium, potassium and creatinine. A special urine collection syringe was used for this purpose.
A saliva sample was obtained from participants aged 16 and over. Saliva samples were collected for analysis of cotinine (a derivative 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.
2.4.1 Laboratory procedures
The Department of Clinical Biochemistry at the RVI acted as the co-ordinating department for the transport of samples to a variety of individual departments within the RVI undertaking analysis for the 2008 Scottish Health Survey as well as ABS Laboratories in the case of saliva 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 and highly abnormal results were immediately reported to the Survey Doctor. Where prior consent had been obtained, the Survey Doctor notified and advised the informant and 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.
Table 2.1 shows reference ranges used for each of the analytes measured in the 2008 Scottish Health Survey. 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). There are no reference ranges for urinary sodium, potassium and creatinine in spot urines, or for saliva cotinine. Table 2.1
2.4.2 Non-fasting blood sample
Measurement of total cholesterol was carried out in the Biochemistry Department at RVI using a Cholesterol Oxidase assay method on an Olympus 640 analyser calibrated to Centre for Disease Control ( CDC) guidelines.
HDL-cholesterol analysis was carried out in the Biochemistry Department at RVI using a direct method (no precipitation) on an Olympus 640 analyser, as it was in 2003. In 1998 the HDL-cholesterol analysis was carried out after PTA precipitation, the 2003 and 2008 data are therefore not directly comparable with those in 1998.
C-reactive Protein ( CRP)
Measurement of CRP was carried out in the Biochemistry Department at RVI using the N Latex CRP mono Immunoassay on the Behring Nephelometer II Analyzer.
The measurement of total glycated haemoglobin (HbA1c) was carried out in the Biochemistry Department at RVI using the Tosoh G7 analyser, which was calibrated using Diabetes Control and Complications Trial ( DCCT) standards.
Fibrinogen analysis was carried out in the Department of Haematology at RVI using the Auto Coagulation lab ( TOP) CTS analyser. The 2003 fibrinogen samples were analysed using the Organon Teknika MDA 180 analyser. However, the methodological principle remains the same. The modification of the Clauss thrombin clotting method was used. Analysis carried out in 2007 for the Healh Survey for England (which uses the same laboratory) showed the correlation between results from the two analysers to be 0.96, therefore the results can be regarded as comparable. 1
2.4.3 Urine sample
Urine sodium, potassium, and creatinine
All urine analytes were assayed on an Olympus 640 analyser. Urine sodium and potassium were analysed using the indirect ISE method. Urine Creatinine was analysed using the Jaffe method.
2.4.4 Saliva sample
Saliva samples received at RVI are checked for correct identification, assigned a laboratory accession number and stored at 4°C. Samples are despatched fortnightly in polythene bags (20 samples per bag) by courier for overnight delivery to ABS Labs. This laboratory specialises in accurate measurement of low levels of cotinine and therefore takes special precautions to ensure that no contamination by environmental tobacco smoke occurs.
Prior to the 2008 study, the analysis method used a specific assay using liquid extraction and gas chromatography with nitrogen phosphorous detection (the technique known as GC-NPD). 2 A new method was introduced for the 2008 study onwards using high performance liquid chromatography coupled to tandem mass spectrometry with multiple reaction monitoring ( LC-MS/MS). 3 The Tomtec Quadra was used with the LC-MS/MS to allow for the automation of some of the sample preparation, where volume of reagents was non-critical; this was validated before use.
To ensure that the LC-MS/MS technique provided results which were comparable with the old GC-NPD2 method, these two techniques were cross-validated. This involved the analysis of previously analysed samples from more than one study using both techniques. As the results obtained for individual samples from both techniques were within ±30% of each other (equivalent to a coefficient of variation of ±15% for mean values, the quality control standard), this showed that both analytical techniques produce similar results. Therefore either method can be used to produce cotinine results 3, and the results from the two methods are interchangeable. In addition the samples supplied from the latest International inter-laboratory study were also analysed on both methods. 3
One benefit of the LC-MS/MS assay is that it is less prone 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. Under the previous method, some saliva samples were too contaminated to allow accurate measurement of low cotinine levels, whereas with LC-MS/MS fewer samples do not yield results. Therefore, once cross-validation was complete, samples from smokers were assayed either by GC-NPD or LC-MS/MS depending on the availability of equipment and the number of samples to be analysed, while samples from non-smokers were analysed solely by LC-MS/MS.
Initially the LC-MS/MS method used a low range of assays from 0.1 to 100ng/mL, with samples for smokers being re-assayed using the original GC-NPD method. Later in the year, a high calibration range was introduced for the LC-MS/MS method, first measuring from 1 to 1,000ng/mL, and later adjusted to measure from 10 to 1,000ng/mL.
Analyses of cotinine were suspended between April and August 2008 while the laboratory moved premises.
2.5 INTERNAL QUALITY CONTROL ( IQC)
2.5.1 Explanation of IQC
The purpose of 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) value 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 ( SD) and the coefficient of variation ( CV) are measures of imprecision and are presented here. Internal QC values are assessed against an acceptable range and samples are re-analysed if any of the Westgard rules have been violated. 4, 5, 6
2.5.2 Non-fasting blood sample
Low, Medium and High control materials are assayed at two hourly intervals. Table 2.2 shows the monthly internal quality control results for total cholesterol. Table 2.2
Table 2.3 shows the monthly internal quality control results for HDL-cholesterol. Table 2.3
Based on materials in use in the department, the Biochemistry department at RVI aim to achieve levels of reproducibility comparable to company literature, with a coefficient of variation ( CV) of <3%. However, realistically the imprecision at the low end of the analytical range leads to a CV of about 6%. Table 2.4 shows the monthly internal quality control results for C-reactive protein. Table 2.4
The analytical methods used for Glycated Haemoglobin measurement in the United Kingdom are required to be traceable to the work carried out on the Diabetes Control and Complications Trial ( DCCT) part of the National Glycohemoglobin Standardisation Program ( NGSP) in the USA. The Secondary Reference Laboratory ( SRL) in the University of Minnesota was the main analytical laboratory for the DCCT work. The internal quality control results for Glycated Haemoglobin are DCCT standardised, and are shown in Table 2.5. Table 2.5
Control plasmas are assayed at regular intervals and instrument function tests are monitored continuously for fibrinogen with the control interval specified as every 4 hours or 100 specimens. Significant deviations from specified limits are flagged and must be acknowledged by the operator. Table 2.6 shows the monthly internal quality control results for two fibrinogen levels. Table 2.6
2.5.3 Urine sample
Sodium, potassium, creatinine
Urine samples were assayed twice a day (am and pm). The IQC results for sodium, potassium, and creatinine are shown in Tables 2.7 to 2.9. Tables 2.7-2.9
2.5.4 Saliva sample
16 non-zero calibration standards were run for each batch of the low range assay (0.1-100ng/ml), and 12 for the high range assay (10-1,000ng/ml). Six Quality Control 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%. A summary of these monthly results for six levels of cotinine is presented in table 2.10. Table 2.10
2.6 EXTERNAL QUALITY ASSESSMENT ( EQA)
2.6.1 Explanation of EQA
The RVI Biochemistry and Haematology Departments and ABS Laboratories both participate in external quality assessment ( EQA) schemes.
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 Coulter Interlaboratory QA programme, National External Quality Assessment Scheme for Haematology, The Cambridge External Quality Assessment Schemes ( EQAS) and the Central Quality Assessment Schemes ( QAS) are all schemes in which the laboratories participate on a routine basis. RIQAS is an EQA scheme run by Randox Laboratories.
Each of the figures presented in Tables 2.11-2.18 corresponds with an individual EQA sample.
2.6.2 Non-fasting blood sample
The Clinical Biochemistry laboratory participates in UKNEQAS and WEQAS schemes. Table 2.11 shows the monthly external quality assessment results for total cholesterol. The target and achieved values are shown. Table 2.11
The Clinical Biochemistry laboratory participates in the WEQAS scheme. Table 2.12 shows the monthly external quality assessment results for HDL-cholesterol. The target and achieved values are shown. Table 2.12
Table 2.13 shows the monthly external quality assessment results for C-reactive protein. The target and achieved values are shown. Table 2.13
Table 2.14 shows the monthly external quality assessment results for glycated haemoglobin. Table 2.14
The Haematology laboratory participates in Central QAS schemes fortnightly. Table 2.15 shows the monthly external quality assessment results for fibrinogen. Table 2.15
2.6.3 Urine sample
The Clinical Biochemistry laboratory participates in the WEQAS scheme for the urine analytes (sodium, potassium, and creatinine). Tables 2.16 to 2.18 show the monthly external quality assessment results for sodium, potassium, and creatinine. Tables 2.16-2.18
2.6.4 Saliva sample
There was no external quality control scheme available in 2008 to analyse cotinine but ABS Laboratories participates in world-wide inter-laboratory split analyses when performed to ensure comparable results.