Controversial aspects of NORIP

NORIP, Malmø 27/4-2004

Controversial aspects of NORIP

Peter FeldingCopenhagen General Practitioners’

Laboratory

Common Nordic Reference Values, Session 15, NFKK 24 – 27 April 2004



• 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


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. 1987 May;25(5):337-42. ** These are the controversial parts in NORIP


How to select the reference sample groups? General aspects*

General population

A posteriori selection:Ideally random recruitment from the general population to form an initialsample group followed by exclusions

A priori selection:Ideally random selection from the reference population or defined subgroups in the reference population

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.

Reference population (unknown) A B


All healthy Nordic adults (reference population)

102 healthy adult subpopulations (around local labs)

3000 healthy Nordic adults (reference sample group)

Selection of the reference individuals in NORIP:

Representative? Random?

Representative? Random?


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.)







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?


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.







Determination of reference values

• 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.

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:









Deviating new reference intervals

Enzymes

Bilirubin

Calcium

Creatinine

Potassium

Change of upper limit


Reference intervals (>= 18 year) Patient results:

Short name (no change)

Long name changed to:

Kode changed to:

old new old = f x New

U/l U/l f= ALAT;P P-Alanine transaminase; cat.c.(IFCC 1998) NPU19651 K: 35 K: 10 - 45 1

M: 50 M: 10 - 70 1 ASAT;P P-Aspartate transaminase; cat.c.(IFCC 1998) NPU19654 K: 35 K: 15 - 35 1

M: 50 M: 15 - 45 1 Creatinkinase;P P-Creatine kinase; cat.c.(IFCC 1998) NPU19656 K: 30 - 150 K: 35 - 210 1

M: 50 - 270 M<50 y: 50 - 400 1 M50 y: 40 - 280 1

Gamma-GT;P P-gamma-Glutamyltransferase (IFCC 1998) NPU19657 K: 60 K < 40 y: 10 – 45 0,94 M: 90 K 40 y: 10 – 75 0,94 M < 40 y: 10 – 80 0,94 M 40 y:15 –115 0,94

Amylase;P P-Amylase; cat.c.(IFCC 1998) NPU19652 70 - 300 25 -120 3,3 Bas.fosfatase;P P-Alkaline phosphatase; cat.c.(IFCC 1998) NPU19655 70y:80 - 275 35 - 105 2,4 LDH;P

P-Lactate dehydrogenase; cat.c.(IFCC 1998)

NPU19658

70y: 100-400 150 - 450

35 - 105 < 70 y: 105 - 205

2,4 2,1

70 y: 115 - 255 2,1 Examples: 1. A new male patient value for ALAT of 70 U/ l is now within the reference interval . The o ld value on the same sample would be 1 x 0,70 = 0,70 U/l and above

the old reference limit. This means fewer pathological values after 11 03 2004.. 2. A new patient value for Bas. fosfatase of 105 U/ l is just within the new reference interval for <70 y. The o ld value on the same sample would be 2,4 x 105 =

252 U/l and well within the old reference interval. This means more patological values after 11 03 2004.

New Nordic reference intervals for enzymes

KPLL introduced the below changes 11 03 2004


The changes:Fewer pathological results for ALAT, CK ,GGT and Amylase More pathological results for Alkaline phosphatase and LDH


Reference intervalsnon-enzymes

Property Reference intervalsNORIP Tietz [14] Laurell [15]

Serum Plasma Serum Serum

Name Unit Gender Age Low High Low High Comment Low High

Comment Low High

Comment18-39 48

40-6936

Albumin g/L FM70 34

45 35 52 >60y: 32-46 36 48

Bilirubin µmol/L FM 18 5 25 5 21 20

Calcium mmol/L FM 18 2.15 2.51 2.15 2.5 >60y: 2.2-2.55 2.20 2.60

18-49 2.6 6.4F 50 3.1 7.9

18-49 3.2Carbamide mmol/L

M 50 3.58.1

2.1 7.1 >60y: 2.9-8.2 3.3 9.7

18-29 2.9 6.1 3.2-3.4 5.6-6.330-49 3.3 6.9 3.4-4.1 6.0-7.2Cholesterol mmol/L FM

50 3.9 7.8

3.7-4.5 6.9-7.9

Each 5-y periodand gender

3.5 8.0Increase

>40y, F>M

F 18 50 90 53 97 45 100

Creatininium µmol/LM 18 60 100

See TABLE XVI and plot ofenzymatic -, Vitros - and Jaffémethods on NORIP home site[2]

62 115 55 115

Glucose mmol/L FM 18 4.0 6.0 4.2 6.3 Fasting (>12 h) 4.1 5.9>60 y: 4.6-6.4, >90y: 4.2-6.7

3.31 5.611fB, 10-15%higher in S/P

F 18 1.0 2.7 0.9-1.0

2.0-2.5 0.75 1.90 HDL-chol mmol/L

M 18 0.8 2.1

0.7-0.8

1.6-1.7

Each 5-y periodand gender.

0.70 1.60


Property Reference intervalsNORIP Tietz [14] Laurell [15]

Serum Plasma Serum Serum

Name Unit Gender Age Low High Low High Comment Low High

Comment Low High

Comment F 9 30.4 10 29

Iron µmol/LM

18 9 34 Results <6 mol/L removed12 31

13 36

18-49 0.10F 50

0.50 0.15 0.50Iron sat.

M 180.15

0.57

Oestrogen users and iron <6mol/L removed

0.20 0.50

18-29 1.2 4.3 1.5-1.8 3.8-4.3

30-49 1.4 4.7 1.8-2.5 4.0-5.2LDL-chol mmol/L FM

50 2.0 5.3

LDL-chol = cholesterol – HDL-chol - triglyceride/2, wheretriglyceride is <4 mmol/L

2.2-2.6 4.8-5.8

Each gender and 5-year period.

2.2 6.2Increase

>40y, F>M

Magnesium mmol/L FM 18 0.71 0.94 0.66 1.07 0.7 1.1 Potassium mmol/L FM 18 3.6 4.6 3.5 4.4 3.5 5.1 3.5 5.0

F 18 0.85 1.50 0.76 1.4118-49 1.65 1.53Phosphate mmol/L

M 500.75

1.350.71

1.23

0.87 1.45F>60y: 0.90-1.32,M>60y: 0.74-1.20

Protein g/L FM 18 62 78 64 79 64 83 >60y: 2 lower

Sodium mmol/L FM 18 137 145 144 136 145 136 146 TIBC µmol/L FM 18 49 83 47 80 Oestrogen users removed 44.8 71.6 46 70

F0.41 -0.59

1.63 –2.62

Triglyceride mmol/L

M

18 0.45 2.60 Fasting (>=12 hours)0.50 -0.62

2.27 –3.70

Gender and age(each decade)partitioning.Increasing with age.

0.4 1.6 Increase

>40 y

18-49 350F 50

155400

120 340Urate µmol/L

M 18 230 480 130 390

F>60y: 200-430,M>60y: 250-470

160 450







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)


Reference intervalsHaematology

1800 ref. individuals from mainly Finland and SwedenComponent

Suggested referenceinterval

Female 7,3 – 9,5B–Hemoglobin (µmol/L)

Male 8,3 – 10,6

Female 0,350 – 0,458B–EVF

Male 0,393 – 0,501

Female 3,94 – 5,16B–Erythrocytes (1012/L)

Male 4,25 – 5,71

Erc–MCV (fL) All 82 – 98

Erc–MCH (fmol) All 1,7 – 2,1

Erc–MCHC (µmol/L) All 19,7 – 22,2

B–Leukocytes (109/L) All 3,5 – 8,8

Female 165 – 387B–Trombocytes (109/L)

Male 145 – 348







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)


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.


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.


Therapeutic intervals

• Are subgroups of decision limits

• Can relate to drug concentration or

• Other properties influenced by drugs or other treatment (INR, HbA1c, Glucose?)


Thank you for your attention

Any questions?

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Controversial aspects of NORIP