Blood tests are used to
The levels of proteins in blood provide a useful means of detecting and diagnosing disease. But, this is only effective if the results of protein measurements are reliable and accurate. A certified reference material, produced by a European team, is now being used globally by both laboratories and equipment manufacturers. Quality assurance surveys show that it has dramatically improved agreement between institutions.
Everyday, thousands of blood tests are performed. Many of these tests measure the concentration of proteins present in patients' blood. Over 50 proteins in the blood circulation have either a protective function or a metabolic role. They are necessary to transport hormones and nutrients around the body and to help the body respond to inflammation and trauma and they assist in the defence against infection.
Disease detection and prevention
For some time, measuring the levels of these proteins has been a means of detecting, diagnosing and monitoring diseases. More recently, plasma protein measurement has been used in preventive medicine. This "biochemical biopsy" is readily accessible, can be repeated without injury to the patient and can yield relevant information which is often unattainable by any other means.
It is crucial that the measurement of plasma proteins is correct. However, ensuring this is more difficult and complex than one would initially imagine. The use of a reference material, which has a known, specific concentration of proteins, can aid the accuracy of measurements. Equipment can be calibrated reliably and the results accurately determined.
Several reference materials have been used world-wide since the mid-70s. The most important of these were the World Health Organisation (WHO) International Reference Preparations and the First United States National Reference Preparation for Specific Human Serum Proteins. Despite their existence, Quality Assessment surveys in Western Europe have shown poor agreement between laboratories and the manufacturers of the equipment needed to make the protein measurements. Patients' results differ from laboratory to laboratory both between and within countries as a result of the commercial calibrants available being different.
The project participants, who are all world leaders in the research and clinical application of plasma protein measurements, were appalled by this catastrophic situation. They knew the consequences of false measurements; patients' health could be jeopardised and research results into the links between disease and plasma proteins become useless if they are not based on the same reference point.
Consequently, a project began to prepare a Certified Reference Material (CRM) for manufacturers and laboratories to verify their equipment is giving correct results for the measurement of 14 plasma proteins. This material would provide a single point of reference for laboratories world-wide.
The European partners were members of a group called the International Federation of Clinical Chemistry Committee on Plasma Proteins. The group also worked with laboratories in the USA and Japan. Each of them brought much knowledge and experience to the project.
The process to develop this important material was not trivial. It had to be very similar to the samples being measured daily in laboratories, as well as being able to last for several years in storage without any change to the concentration of all 14 proteins. Tests showed that even at 45°C, the protein levels remain constant for at least a year. The partners showed that a number of different techniques, including all those commonly in use, could be reliably calibrated using the material.
From blood donation to the final product
To produce the 40,000 vials of reference material, hundreds of healthy people in five European cities, each donated blood. Details about the volunteers, such as country of origin, race, gender, age, weight and blood group, were recorded in order to separate donor pools of people with similar blood. Any blood containing HIV and hepatitis or other abnormalities was excluded. The blood was processed, preserved and frozen before it was shipped to the central processing centre in Marburg, Germany. The blood was thawed and pooled together. The fats were removed and the protein levels and pH were adjusted. It was filtered in a sterile environment and the vials were filled to 1.00 ml before being freeze-dried and sealed.
All samples produced have exactly the same composition. To measure the protein concentrations precisely, some pure proteins were produced and existing WHO reference materials for plasma proteins were used. The resulting CRM exists as an international master calibrator. It has two to five years life but based on current demand, there are enough samples to last at least ten years. In turn, the CRM is used to develop calibrators for national laboratories as well as commercial calibrants with one to two years of life. The measurements performed with equipment calibrated using these are reliable.
At the time of the QA survey, results varied by as much as 50 to 100%. The aim was to reduce this to 5%. Recent surveys show the divergence decreased dramatically once the CRM was introduced. The CRM has also been approved by the Food and Drug Administration in the USA and is distributed by the College of American Pathologists, who are the major providers of plasma protein reference materials in the USA.
The project co-ordinator, Prof John Whicher of the University of Leeds, explains the benefits, "Labs and manufacturers world-wide are now using CRM 470 and the problems of poor agreement in protein measurement are largely solved. Patient and research results can now be confidently transferred between institutions and companies are able to sell diagnostic kits world-wide without major problems of local standards not agreeing. In particular, this allows European companies to sell their equipment in the USA."
He goes on to point out, "As well as these advantages, EC in vitro Directives are
supported. There are also enormous benefits gained through laboratory accreditation and quality assurance as the ability to use target values for external quality assurance values is not possible without agreed standards."
The work continues...
The project partners are continuing the good work. A second project has certified the reference material for a further three proteins and it plans to assign values for other proteins in the future. The group hopes to produce further reference materials.