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An essential part of clinical chemistry is to assess properties in blood samples for diagnosis or monitoring of disease. The knowledge of the limits for these properties in healthy individuals is necessary for the distinction between health and disease. The objective of this project is to determine such limits better than before and to encourage the use of common limits in the Nordic countries when possible.

The project involves the properties of serum most commonly measured in hospital laboratories (serum is the liquid between the blood cells after blood coagulation)

In the project the samples from the healthy individuals are measured together with reference material (pooled unmodified frozen serum from many blood donors) at the participating laboratories. The reference material has previously been analysed with extremely good analytical methods (reference methods). The values from the reference individuals are therefore analytical traceable to the reference material and the reference methods. This is a very important quality of the design of the project.

Reference methods can secure that results of future investigations can be properly compared to the values obtained in this project. However, reference methods are very expensive to use. Therefore the reference material from this project will be kept frozen at - 80°C and be offered for use in similar future project here (next generation?) or in other countries. In this way the values obtained in different project can be properly compared without use of reference methods in each project. Also samples from the reference individuals will be kept frozen at –80°C, which means that the list of properties rather easy can be expanded later with the same traceability to reference material and individuals.

In addition to serum and plasma from the reference individuals, which materials shall be used as described above, also blood will be collected and kept for future use. It will therefore be possible to correlate genotypes (from analysis of DNA) to measured concentrations of components in plasma and serum.

Where it is biological and technical possible the project will support the use of common limits in the Nordic countries (and therefore also within each country). The previous lack of harmonisation is mainly due to lack of knowledge of the true limits, which the project will produce. Common limits will have a great impact on learning and communication within the health systems.

The project is decentralised mainly for practical and economical reasons. Each of hopefully about 200 laboratories in the nordic countries measures according to a common protocol with its routine methods on local healthy individuals and on the reference material. The results from the reference material are used as a common divisor to tie together results from different methods. The decentralised design however also imply, that the future users of the results understand how they were produced and how their own methods are related to them. We expect this to have a psychological and real impact one the acceptance of the common limits.

Objective *

The objective of this project is to established traceable Nordic reference intervals for concentrations of (preliminary) the following components in serum and Li-heparin plasma, altogether 25 components:

*Alanine aminotransferase
*Albumin
Amylase
Pancreatic amylase
Aspartate aminotransferase
*Alkaline phosphatase
*Bilirubin
*Calcium
Urea
*Cholesterol
HDL-cholesterol
*Glucose
*Creatinine
*Creatininekinase
*gamma-Glutamyltransferase
Iron
Lactatedehydrogenase
*Magnesium
*Potassium-ion
*Sodium-ion
*Protein
*Phosphate
TIBC
*Triglycerides
*Urate

The list may be expanded during the project or later.

The reference intervals are for healthy adults with blood sampling being performed in the sitting position. More than 25% will be taken from fasting persons in the morning.

In the Nordic countries analyses of the components above amount to about 30 - 40 % of all analytical work done within clinical biochemistry in hospital laboratories and covers the analytes most commonly monitored in serum.

A sample of calibration material (CAL) (10) will be analysed in the same analytical runs as the samples from reference individuals and thus the analytical results will be linked to concentrations of the respective components in a primary calibrator. CAL has already been issued with target values of 18 analytical components, obtained by reference methodology (components indicated by * and Thyroxin as well). The project's organisers will issue given concentration values for the remaining components in the CAL calibrator.

The project will establish a biobank for storing data and samples from reference individuals as well as reference material (controls and calibrators):

All data from the project (method data, anonymous personal information, along with anonymous analytical results) shall be accumulated in a database, which will be accessible via Internet.

Reference material and anonymous but numbered serum, heparin plasma and EDTA-blood samples from reference individuals will be kept at - 80 °C. This will make it possible with the same traceability to:

decide if new analytical methods for the same analyte can use the reference intervals already established (using the reference material)
compare the results of the project with similar future studies here or in other countries (the reference material can either be sold or donated/exchanged)
establish reference intervals for other quantities than studied in this project but traceable to the same reference individuals and CAL (other analyses, same reference individuals and the same reference material)
correlate genotypes to concentrations

It will be possible to set reference target values on the stored CAL that can then replace

the given values used. The reference intervals that are tied to the given values for components

of CAL can then easily be replaced by calculations to fully traceable reference intervals.

The project shall identify (among the participating methods) those methods that directly or after recalibration can use the same reference intervals for a given group of individuals.

The project shall define such groups of individuals. (The reference individuals will be characterised by sex, age, weight, height, day of menstrual cyclus (women), geographical area, ethnic origin, heredity for diabetes and smoking, drinking, exercise and medication habits.)

In the project also blood sampling conditions that can share common reference intervals will be defined (time of blood sampling in relation to month, weekday, time of day and time interval from last meal).

Background *

The development of clinical biochemical methods has steadily been moving towards automation and use of commercial kits in fewer types of analytical instruments. The use of common international and Nordic quality control material has also been steadily increasing. This has meant a more even analytical quality in the Nordic countries and a better analytical traceability. The reference intervals in use have lagged behind this development and they are neither traceable nor in many instances relevant. Of course there are exceptions, but many laboratories have combined data from the kit producers with the most relevant data found in the literature, their own former reference intervals (that may have been transformed because of method change) as well as the reference intervals from laboratories close by, to form their own reference intervals presently in use. These constructed reference intervals are at best then tried out by measuring a limited number of blood donors or samples drawn from the laboratory workers or selected patients. Characteristically therefore a laboratory does not know whether or not the used reference intervals reflects the concentrations in the surrounding healthy population when measured with the method in use.

The reason for this discrepancy is that individual laboratories do not have the financial resources or manpower, to set their own traceable and correctly worked out reference intervals.

In national and Nordic quality assessments surveys a great variation in the reference intervals used by different laboratories has been reported although the analytical results from pooled frozen normal human sera have been in good agreement. A difference in width of the reference intervals as well as in the level was revealed. The analytical results in the surveys were expected to represent an average value, but they were found lying in some laboratories at the upper and in others at the lower end of the reference intervals in use (communications during DEKS-meetings from P. Rustad, Norway; H.H. Dalsager, Denmark; E. Olafsdottir, Iceland). It is highly unlikely that this can be explained by true differences in the concentration of analytes in the population served by each laboratory. It is reasonable to assume that most analytical methods for the same quantity can give the same analytical results for a normal person if these methods are properly calibrated. Thus it should be possible to use common reference intervals in a much higher degree in the Nordic country than is practised today. The laboratories are therefore urged to join forces and put this project into realisation. In a large project like the present one, experts in the field will be involved and it will be possible to follow approved recommendations or otherwise work out variations from those procedures in a well founded way (1 - 7).

For the last several years heparinised plasma has increasingly been replacing serum as the chosen matrix. It was therefore decided to draw a certain proportion of the samples as heparinised plasma.

The above problems concerning reference intervals are not unique for the Nordic countries. In the USA a recent study showed a considerable difference between used reference intervals and the results obtained when samples from healthy individuals were analysed in a particular laboratory (8).

Before the initiation of the Nordic Protein Project (9) the project leaders knew that poor standardisation of methods and inadequately worked out reference intervals for the most commonly analysed plasma proteins were less acknowledged and more neglected than is the case with the analytes in the presently proposed study. The Nordic Protein Project had a great impact in establishing order and traceability into the analytical practice of specific proteins and it has been the leading model for the design of the present project.

Organisation and time schedule *

The project is organised by a steering group, appointed by the national societies in clinical chemistry in the Nordic countries.

The project will be carried out during 1999 as part of an extended distribution of external quality assurance material from one or more of the Nordic External Quality Assurance Organisations (Labquality (Finland), Equalis (Sweden), DEKS (Denmark), NKK (Norway)).

The project organisers will invite (urge) the following numbers of laboratories to participate: about 40 in Denmark, about 60 in Sweden, about 40 in Norway, about 40 in Finland and 3 in Iceland. All of these, with the exception of Sweden, participate in Labquality's FHK quality assurance program.

After the project's completion, the material kept in the established biobank will be administrated by DEKS but be the property of the Nordic Society for Clinical Chemistry (Nordisk forening for klinisk kemi), which also will hold the copy right of the data in the biobank (database).

Reference individuals *

Criteria for inclusion *

The reference individual should

be feeling subjectively well
have reached the age of 18
not be pregnant or breast-feeding
not been an in-patient in a hospital nor been subjectively dangerously ill during the last month
not had more than 2 measures of alcohol (24 g) in the last 24 hours
not given blood as a donor in the last five months
not taken prescribed drugs other than the P-pill or estrogens (female sex hormone).
during the last two weeks
not smoked in the last hour prior to blood sampling

Criteria for exclusion *

Plasma- or serum-Glucose over 11.0 mmol/L

Other analytical results that clearly point to a disease (exclusion means that results of all analytical components are to be excluded).

Data recorded *

age
sex
height
weight
for women: Date of 1. day of last menstrual period
ethnic origin (half or more of the genetic origin)
heredity for diabetes
number of years residing in a Nordic country
name of a disease(s) that requires continuous monitoring or treatment by a doctor
any medication (the P-pill and iron tablets included)
strenuous exercise during the last week
habitual alcohol consumption of 0, 1-21 or more than 21 measures of alcohol per week
habitual smoking of 0, 1-5 or more than 5 cigarettes/cigars/pipes per day
number of hours from the last meal before blood sampling
date of blood sampling (day in month, month, hour, day of week)
number of total blood donations to a blood bank

Recruitment *

Reference individuals can be recruited from the laboratory personnel and their relatives or acquaintances, from new blood donors or ones that seldom donate blood. Equal sex and age distribution should be sought according to the protocol.

Payment *

The participants will not receive any financial compensation. The local laboratory may use their own resources, should they wish to compensate for time lost from work or for transportation expenses.

Sampling procedure *

A maximum of 50 ml blood will be drawn from a cubital vein by laboratory technicians or nurses. This is about one tenth of the usual amount donated by blood donors in blood banks (If the samples are taken in connection with blood donation, the rules about maximum allowed blood volume will be kept). The blood sampling is considered harmless and the inconvenience is limited (the same as experienced after a routine blood sampling from a vein in the arm). The samples will be drawn at the sampling locations of the laboratories, in blood banks or in the homes of the reference individuals.

Assurance *

Will depend on the country and laboratory. The reference persons shall have the same rights as patients.

Ethics *

The data will be made anonymous in the following way:

All data concerning the reference individual will be linked to a running project number. Name and "cpr.nr." will not be linked to the project number or any other data in the project. The reference individual will however sign an informed consent. No other person-related information is on the consent form (neither the running project number nor any other information that can link the name with any data). The reference individual will get his/her project number with the information about the project. Only the reference individual (or whoever he gives the information to) will know the given number is linked to that particular individual. Each reference individual will have an opportunity to contact the laboratory doctor and get the local analytical results linked to one issued number. The laboratory cannot trace the results from a sample number to the reference individual and therefore cannot let a person know if serious pathological results are found.

Informed consent *

The primarily selected reference individuals will in proper time before the sampling receive the following information and the questionnaire:

Nordic Reference Intervals in Clinical Chemistry

Information to participant

Dear Reference Individual.

Results from laboratory investigations are used by doctors to diagnose disease. The results from several common laboratory investigations do not distinguish easily between healthy and diseased individuals, i.e. we do not know with certainty what is normal. This is why the Nordic hospital laboratories have started a joint project in order to establish reference ranges for common blood analyses in the Nordic countries.

If you are 18 years of age or older and are feeling healthy, you are kindly asked to participate in this project as a reference individual by:

Donating a blood sample (about 50 ml which is about one tenth of the usual amount donated by blood donors in blood banks).
Giving permission to do the analyses performed in the hospital laboratory on your anonymous sample including genotypes (DNA-types) of relevance for the measured concentrations.
Giving permission to store the anonymous samples for later repetition of similar analyses by the laboratories.
Answering the enclosed questionnaire.

The results will be made anonymous for everyone except you in the following way:

At the time of blood sampling you will be given a sample number that will be used for labelling the samples.

Information from the questionnaire as well as the analytical results will be tied to that sample number. Your name and "cpr.nr." will not be linked to the sample number (and we do not wish to have the "cpr.nr."). In that way you will be the only person that can link yourself to the sample number.

As we do not have your name or "cpr.nr." linked to analytical data, we cannot send you the laboratory results, even if we desired to do so.

The analytical results from all samples in the project will be accessible on the Internet identified by the sample number. Each participant can also contact the blood sampling laboratory and ask for the results should he/she wish to do so. The sample number must then be used as means of identification. If you don't want anyone to know which sample you have donated you should destroy your sample number.

Further information will be given by the doctor in charge: ______________________________

Tel.: _____________________

The blood sampling is done by usual sampling procedures from a vein in the arm. This is considered completely harmless. In a few instances you may get a blue mark around the point of puncture and a slight discomfort that should disappears within a short time. It will be your own choice to participate and you may withdraw your participation until you leave the laboratory after blood sampling.

By signing this document you have accepted to participate in the project.

_________________________________________________

Signature of the participant

This form should be signed in duplicate, one copy for the participant and one for the laboratory.

To the medical technologist:

The sample number should not be put on the form that is retained at the laboratory."

Questionnaire *

The reference individuals must answer the following questionnaire.

The questionnaire together with the above information will be presented for the primarily chosen individuals in proper time before the sampling procedure. The selected reference individuals should be familiarised with the questionnaire before coming for blood sampling. They should complete the questionnaire as fully as possible and refrain from coming if the required criteria are not met. Any unanswered questions will be completed with the assistance of a medical technologist or nurse at the time of blood sampling.

Nordic Reference Intervals in Clinical Chemistry

Questionnaire to participant

Sample number __________________ (to be filled out by nurse)

You cannot participate in this investigation if:

You are feeling unwell today.
You have been an in-patient in a hospital or if you have been dangerously ill (according to your own judgement) during the last month.
You have had more than 2 measures of alcohol in the last 24 hours (a measure is equal to 12 g alcohol, the equivalent of the amount of alcohol found in one pilsner beer or one glass of wine).
You have given blood as a donor in the last five months.
You are pregnant or are breast-feeding.
You have taken prescribed drugs other than the P-pill during the last two weeks.

If you are a smoker you must refrain from smoking one hour prior to the blood sampling.

Questions to be answered by the participant:

Age: _________ years Sex: o female o male

Height: _________ cm Weight _________ kg

For women: Date of 1.day of last menstrual period:

Day of month:________ month:_____ year :_______

(May not be answered if irrelevant)

Ethnic origin ________________________________________ (If you think your genetic origin is half Nordic or more please write "Norden" otherwise write the country/ies which the major part of your genetic heritage came from).

How many years have you lived in your present country of residence? ________

Do you normally smoke (mark with one x):

o 0 cigarettes/cigars/pipes per day?

o 1-5 cigarettes/cigars/pipes per day ?

o more than 5 cigarettes/cigars/pipes per day?

Do you normally drink (mark with one x):

o 0 measures of alcohol per week ?

o 1-21 measures of alcohol per week ?

o more than 21 measures of alcohol per week ?

Yes No

o o Does any of your siblings or parents have diabetes or did they have when alive

o o Have you been diagnosed with a disease that requires continuous monitoring or treatment by a doctor or in a hospital ?

If yes, kindly specify the disease:

_______________________________

(disease)

Yes No

o o Have you taken the P-pill or estrogen preparations (female sex hormones) during the last month?

o o During the last week, have you taken any other medication (incuding iron tablets) than the ones mentioned above ?

If yes to one or both of the above two questions, write the name(s) of the medication(s):

_____________________________________________

o o Have you participated in strenuous sports during the last week (e.g. run more than 10 km in one go, trained in fighting sports, like karate, boxing or equivalent or been active in body building)?

To be filled out with the medical technologist (or nurse):

Number of hours from the last meal before blood sampling: _________ hours

To the medical technologist (or nurse):

Time of sampling:

Weekday 1 - 7 (Monday = 1 ...... Sunday = 7):______________

Day of month (1-31):_____

Month 1 - 12 (January = 1 .... December = 12):______________

Time of the day (00.00 till 24.00):______________

Identification of the laboratory (Labquality number):______________

The sample number, which is used for identifying the samples taken from the reference individual who has filled this form, shall be written or labelled on the form and samples. The name of the individual or the "cpr. nr." must not be written on the form or any of the samples.

Materials *

Samples from reference individuals *

The participating laboratories will collect and freeze serum samples, each from 25 - 50 reference individuals, according to a written protocol. Li-heparin plasma will be collected from approximately 10 % of the reference individuals and EDTA-buffy coat from all individuals.

All reference samples below shall be kept frozen until analysed (-20 °C for one month or -80 °C).

In an effort also to get valid reference intervals for Li-heparin plasma, it is, as an option, also allowed to analyse fresh Li-heparin plasma samples according to protocol mentioned below (see DESIGN).

Each laboratory will receive a series of running project numbers (one number for each person) for labelling the samples as well as to record the analytical results by. The laboratories will also receive the transport tubes and other devices for mailing of frozen samples to the "Biobank".

Reference material *

The participating laboratories will receive the following liquid reference material in frozen form either from Labquality or the national quality assurance organisation. The reference material shall be kept frozen until analysed (-20 °C for one month or -80 °C).

• CAL (pool of unmodified serum from healthy donors, with the concentration of 18 components determined by reference methods)(10).

• Donor serum pool, concentrated by removing water by freezing; HIGH (11).

• Donor serum pool, diluted from HIGH by adding one part of NaCl/CaCl₂ solution; LOW

• Serum pool from donors on the P-pill; P

• X (prepared in the same way as CAL).

Analytical methods *

The laboratories shall use their routine methods. However only Vitros and "IFCC 37 °C" methods shall be used for enzymes. Total iron binding capacity (TIBC) can be measured with immunochemical methods for transferrin or iron binding methods.

Design *

The participating laboratories will measure as many of the 25 components as possible.

Thawed materials: Along with the thawed samples from the reference individuals the above listed reference material will be analysed for each component in the same analytical run. CAL is to be measured 10 times, placed evenly between samples in the analytical run for each component. The other control material is to be measured 3 times, at the beginning of the run, in the centre and at the end of each run.

Fresh materials (option): Only X is to be measured 10 times evenly distributed between samples from reference individuals in each series of fresh materials.

The reference samples should be analysed in the same way as patient samples following the same working procedures.

The reported results should be validated in the same way as the laboratory practices in routine work, so the laboratories usual controls must also be included in the run.

7 serum samples, and 1 "buffy coat" sample (from EDTA sample tube) from each reference individual and 2 Li-heparin plasma samples from 10% of the individuals are to be sent to a central storage site. Finally this will be DEKS. There they will be kept at - 80 °C until later use.

It is allowed locally to organise that samples collected at one laboratory could be analysed for some or all components at another laboratory (both the sampling laboratory and the analysing laboratory will be known from the report for every sample and analysis)

The following information must be sent to a central committee either on paper, a spreadsheet or directly via Internet to a database that will be connected to the ROSAN database.

• Method data (from Labqualities method description codes)

• Analytical results of the samples from the reference individuals identified by the running

numbers and results from the supplied material described above

• Data from the reference individuals, sampling date and sample preparation also linked to the running number.

The central group will transform all analytical data by multiplication with a factor CAL-target/ CAL-local. CAL-local is the mean of the local results for each component in the run after outliers have been rejected. This means that CAL is used as a calibrator for transforming the results. The organising committee will decide how given values will be set for the components that lack a target value (e.g. the mean of results from accepted methods after outliers have been rejected).

The transformed results from control analyses will be evaluated from the ratio High/Low and the level of P (12).

Based on this evaluation it will be decided whether the method (or possibly the Labquality method group) should be investigated further by selected laboratories, where a limited number of reference samples with high and low values from the respective run (or from other runs in the method group) will be reanalysed. Samples from the biobank can be used for this purpose. Following such a study, it will be decided for each component, which methods can use common reference intervals after a given transformation of results. All transformed results from these methods will be pooled to establish common reference intervals for the relevant groups of individuals.

The established reference intervals are linked to target values or given values for CAL and should only be used in laboratories that can reach the results within a specified target limit around the target value or given value of CAL or X. This target limit will be set in agreement with the quality specifications necessary for the use of common reference intervals (12). If this is not possible the reference values can be transformed according to the local results for CAL to be used locally.

Statistical methods *

The reference limits will be calculated as 2.5 and 97.5 percentiles of the population of accepted reference values according to IFCC recommendations. Only in subgroups parametric methods may be necessary, but the nature of this will depend on the results and the more detailed purpose (6).

Economy *

The project will be financed mostly by the participating laboratories. This is done by the laboratory paying for participation in the project (the cost will be equivalent to a one-year participation in a FHK program which is about 3500 DKr), in return it will receive a special delivery of the control and calibration material from Labquality and DEKS. The work performed in collecting and treating samples and performing analyses will also go on the laboratories' budget.

The Nordic Societies in Clinical Chemistry have supported financially the meetings in preparation of this project. Financial contributions from public foundations (sources) will be sought to cover expenses for production and storage of reference material, administration and database facilities. Also support from kit producers will be sought.

Literature *

Solberg HE (1987). Approved Recommendation (1986) on the Theory of Reference Values. Part 1. The Concept of Reference Values. Clin Chim Acta 165:111-118; J Clin Chem Clin Biochem 25:337-342; Ann Biol Clin 45: 237-241; Labmedica 4:27-31.
PetitClerc C, Solberg HE (1987). Approved Recommendation (1987) on the Theory of Reference Values. Part 2. Selection of Individuals for the Production of Reference Values J Clin Chem Clin Biochem 25: 639-644; Clin Chim Acta 170: S3-S12.
Solberg HE, PetitClerc C (1988). Approved Recommendation (1988) on the Theory of Reference Values. Part 3. Preparation of individuals and Collection of Specimens for the Production of Reference Values. J Clin Chem Clin Biochem 26: 593-598; Clin Chim Acta 177: S1-S12.
Alström T et al. Establishing reference values from adults: recommendation on procedure for the preparation of individuals, collection of blood, and handling and storage of specimens. Scand J Clin Lab Invest 1993; 53:649-652.
Solberg HE (1991). IFCC Recommendation - theory of reference values. Part 4. Control of analytical variation in the production, transfer and application of reference values. Clin Chim Acta 202; S5-S12.
Solberg HE (1987). Approved Recommendation (1987) on the Theory of Reference Values. Part 5. Statistical Treatment of Collected Reference Values. Determination of Reference Limits. J Clin Chem Clin Biochem 25: 645-656; Clin chim Acta 170: S13-S32.
Dybkær R, Solberg HE (1987). Approved Recommendation (1987) on the Theory of Reference Values. Part 6. Presentation of observed Values Related to Reference Values. J Clin Chem Clin Biochem 25:657-662; Clin chim Acta170: S33-S42; Labmedica (1988) 5:27-30.
Mold JW, Aspy CB, Blick KE, Lawler FH. The determination and interpretation of reference intervals for multichannel serum chemistry tests. J Fam Pract 1998 Mar;46(3); 233-241.
Hyltoft-Petersen P, Blaabjerg O, Irjala K eds. Assessing Quality in Measurements of Plasma Proteins, The Nordic Protein Project and Related Projects, Nordkem, Nordic Clinical Chemistry Project, Helsinki, Finland, 1994
Uldall A, Steensland H, Nordberg U-R, Loikkanen M, Siekmann L, Schumann G. EQAnews 1998, 9 (3) :39-42.
Letellier G & Desjarlais F. A simple procedure for the preparation of concentrated sera. Clin Biochem1985;18:267-71
Gowans EMS, Hyltoft Petersen P, Blaabjerg O, Hørder M. Analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area.Scand J Clin Lab Invest 1988;48:757-64