1. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Peter Felding
Copenhagen General Practitioners’ Laboratory
Common Nordic Reference Values, Session 15, NFKK 24 – 27 April 2004

2. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Selection of reference individuals
The sample material
The analytical measurements and the correction with CAL Target /Cal measured
Deviating new reference intervals
Haematology -use of EDTA in different forms
Reference intervals- not decision limits
Thank you to the Congress to Pål and to you for this opportunity to discuss controversial aspects of the Nordic reference interval project.
In this projects many decisions have been made and some of these could be controversial. From our own minds and debate the last months we have selected these 6 issues but you are free to ask questions about others. I have marked with red the two most fundamental for the acceptance of the final reference values.
Before we go into details I will remind you of same basic concepts of reference values

3. The concept of reference values*
NORIP, Malmø 27/4-2004
The concept of reference values*
Reference individuals constitutes a
Reference population from which is selected ** a
Reference sample group on which are determined **
Reference values on which is observed a
Reference distribution on which are calculated
Reference limits that may define
Reference intervals (with which the patient result can be compared)
*From J Clin Chem Clin Biochem May;25(5): ** These are the controversial parts in NORIP
A series of papers from IFCC in the 80ies heavily influenced by Nordic colleagues covered this area. This slide is more or less taken from the first paper, which dealt with the concepts and nomenclature. When you look at a patient result you want to compare it with something. In our case this ideally should be results from healthy individuals, who except from fulfilling certain health criteria are similar to the patient. Such individuals are called reference individuals and have to be defined. You can use the shown procedure to create health associated (or other) reference intervals with which you can compare the patient result. This seems ease -but it is not when it comes to practice.
Again you can se, that I have marked the most controversial parts,and we now take the fist: The selection of the reference individuals to the reference sample group

4. How to select the reference sample groups? General aspects*
NORIP, Malmø 27/4-2004
How to select the reference sample groups? General aspects*
General population
Reference population (unknown) A B
A posteriori selection:
Ideally random recruitment from
the general population to form an initial
sample group followed by exclusions
A priori selection:
Ideally random selection from the reference population or defined subgroups in the reference population
The second of the mentioned papers describes how to select reference individuals. It shows two ways a posteriori and a priori selection.
The A procedure was theoretically possible in NORIP however it was not practical possible, and the experience with this method is that only a smaller part of the initial random selected group will volunteer in such studies giving a major problem for the representativeness.
Reference sample group/subgroups
*Inspired by Clinica Chemica Acta,170 (1987) s3-s12
The final reference sample subgroups (after exclusions and partitioning) should represent the corresponding subgroups in the reference population. The first selection of individuals from the general population (A) or from the reference population (B) therefore ideally should be random and all selected individuals used as reference sample group or for further random or total selection to subgroups according to criteria for exclusions or partitioning. NORIP deviated from both these procedures.

5. Selection of the reference individuals in NORIP:
NORIP, Malmø 27/4-2004
Selection of the reference individuals in NORIP:
All healthy Nordic adults (reference population)
Representative? Random?
102 healthy adult subpopulations (around local labs)
Representative? Random?
Note: Within the 102 sub-populations around the lab, the persons with some relation to the lab have higher chance to selected.This could mean an overrepresentation of the middle class or certain ways of living.
3000 healthy Nordic adults (reference sample group)

6. The reference sample groups in NORIP is characterised by:
NORIP, Malmø 27/4-2004
The reference sample groups in NORIP is characterised by:
Recruitment (more or less random) from 102 assumed healthy sub-populations somehow connected to participating laboratories in 5 countries.(Questions: Does the sum of these sub-populations fairly represent all healthy adults in Norden? And how random is the selection from each sub-population?)
Apriori fixed even relative distribution of individuals in 8 predetermined groups defined by age and gender. (Raise the problem: the selected individuals in the predetermined age-subgroups could theoretically for all relevant properties be composed as the corresponding age groups in the reference population, but the sum of the reference sample subgroups (all selected individuals) or later determined reference sample subgroups other than the predetermined could not because the age distribution in these later created reference sample groups does not reflect the age distribution in the corresponding reference populations.)

7. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Selection of reference individuals
The sample material
The analytical measurements and the correction with CAL Target /Cal measured
Deviating new reference intervals
Haematology -use of EDTA in different forms
Reference intervals- not decision limits

8. NORIP, Malmø 27/4-2004
The sample material in NORIP (The preparation of the individuals and the sampling procedure are not considered controversial)
Frozen serum (all laboratories)
Fresh serum (optional)
Frozen Li-Hep plasma (10% of samples at all laboratories)
Fresh Li-Hep plasma (optional)
EDTA blood/buffy coat
(No gel)
The control materials were fresh liquid frozen pooled unmodified
or modified normal serum
Question: Can reference intervals based on frozen material be
used for fresh routine samples? What about plasma contra serum? .

9. The sample material in NORIP continued
NORIP, Malmø 27/4-2004
The sample material in NORIP continued
The raised questions were apriori considered controversial but the use of fresh frozen serum was considered necessary in the chosen design.
As it turned out there seemed to be no major problems connected to the use of fresh frozen serum samples including use of results from this material as reference values for patient results from fresh serum or plasma.

10. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Selection of reference individuals
The sample material
The analytical measurements and the correction with CAL Target /Cal measured
Deviating new reference intervals
Haematology -use of EDTA in different forms
Reference intervals- not decision limits
We wanted reference values as true as possible although we knew that the reference intervals should be used as comparison for less true routine results.
Both the wish for true reference values and the way we tried to obtain these are controversial.
What did we do?

11. Determination of reference values
NORIP, Malmø 27/4-2004
Determination of reference values
Every initial measured result was produced by routine methods and for non enzymes then corrected with CAL Target/mean of CAL measured in the same series. This gives the corrected values high trueness at the mid reference interval level, but:
Eliminates the normally included between series analytical variation (this variation can be added locally to the recommended reference interval) and
May be a problem for the used routine methods, for which the initial measured value does not vary proportional with the true value (e.g. some or all Jaffé methods for Creatinine). The use of high and low controls (modified serum) could not totally eliminate or detect this problem, because only the unmodified controls can be considered fully commutable with patient samples.

12. NORIP, Malmø 27/4-2004
A piece of art from Pål

13. NORIP, Malmø 27/4-2004
Assuming the linear relation with positive Y-intercept the figure shows that in the Jaffé reference values obtained with proportional correction would give narrower reference intervals than true values. And this was also what we found (using results from enzyme methods or HPLC methods as true values)

14. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Selection of reference individuals
The sample material
The analytical measurements and the correction with CAL Target /Cal measured
Deviating new reference intervals
Haematology -use of EDTA in different forms
Reference intervals- not decision limits

15. Deviating new reference intervals
NORIP, Malmø 27/4-2004
Deviating new reference intervals
Change of upper limit
Enzymes
Bilirubin
Calcium
Creatinine
Potassium

16. New Nordic reference intervals for enzymes
NORIP, Malmø 27/4-2004
New Nordic reference intervals for enzymes
KPLL introduced the below changes
The changes:
Fewer pathological results for ALAT, CK ,GGT and Amylase
More pathological results for Alkaline phosphatase and LDH

17. Fewer pathological results for ALAT, CK ,GGT and Amylase
NORIP, Malmø 27/4-2004
The changes:
Fewer pathological results for ALAT, CK ,GGT and Amylase
More pathological results for Alkaline phosphatase and LDH

18. Reference intervals non-enzymes
NORIP, Malmø 27/4-2004
Reference intervals non-enzymes

19. NORIP, Malmø 27/4-2004

20. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Selection of reference individuals
The sample material
The analytical measurements and the correction with CAL Target /Cal measured
Deviating new reference intervals
Haematology -use of EDTA in different forms
Reference intervals- not decision limits

21. NORIP, Malmø 27/4-2004
Haematology
Not all laboratories participated (most of the reference individuals were from Sweden and Finland)
No common control/calibrator
Correction for dilutions due to EDTA in different forms. Dry form recommended.
Influence of tobacco (Lkc and other), age (Hb?), gender (Trc?)
Number of decimals (EVF)
However, the final values seem uncontroversial
(Upper limit 8,8 10E9/l for Leukocytes may be an exception.
10 or 11 10E9/l are presently more used)

22. NORIP, Malmø 27/4-2004
Reference intervals Haematology 1800 ref. individuals from mainly Finland and Sweden

23. Controversial aspects of NORIP
NORIP, Malmø 27/4-2004
Controversial aspects of NORIP
Selection of reference individuals
The sample material
The analytical measurements and the correction with CAL Target /Cal measured
Deviating new reference intervals
Haematology -use of EDTA in different forms
Reference intervals- not decision limits

24. Health related reference intervals
NORIP, Malmø 27/4-2004
Health related reference intervals
Reference intervals describe the distribution of results from healthy individuals. They usually include 95% of these results.
They are created within the profession of clinical chemistry.(The traditional “owner” and decider has been the local laboratory because the intervals to some degree has been considered method specific)
They are still the most frequently used reference for patient results. In particular for properties requested with more than one indication (or with more than one decision limit)

25. Medical decision limits
NORIP, Malmø 27/4-2004
Medical decision limits
Are created using patients and often? common for all analytical methods.
Are used to allocate patients to diagnosis, other investigations, treatments, change of treatment, public support, groups of prognosis etc.
Can be inside or outside the corresponding health related reference interval.
The same property can have several decision limits (because of use in different clinical situations or because of different recommendations).
They typically change with more knowledge without change in measured properties or patients (cholesterol!)
To use them the clinical situation must often be know
The medical societies and the clinicians are the “owners” or deciders.

26. Reference intervals as decision limits
NORIP, Malmø 27/4-2004
Reference intervals as decision limits
Any “test” using a reference interval limit as diagnostic decision limit has a specificity of 95% (or 97,5%) for any disease i.e. the proportion of healthy persons with a ”healthy” test result is 95% (or 97,5%).
The corresponding sensitivities of the test for a variety of diseases are in many cases known but might not be relevant because the relevant diagnostic decision limit probably is not a reference interval limit.

27. Therapeutic intervals
NORIP, Malmø 27/4-2004
Therapeutic intervals
Are subgroups of decision limits
Can relate to drug concentration or
Other properties influenced by drugs or other treatment (INR, HbA1c, Glucose?)

28. Thank you for your attention Any questions?
NORIP, Malmø 27/4-2004
End of show
Thank you for your attention
Any questions?