Dr Bill Bartlett Blood Sciences, Ninewells Hospital & Medical School, NHS Tayside, Scotland, UK.

Transcription

1 Dr Bill Bartlett Blood Sciences, Ninewells Hospital & Medical School, NHS Tayside, Scotland, UK.

2 Biological variation affects the clinical utility of reference values. An understanding of the nature of biological variation will:- enable production of relevant reference values enable effective application of clinical laboratory measurements We should consider additionally qualifying reference values by description of associated indices of biological variation.

3

4 25 Year old Male Patient attending a GP surgery: - Sodium 140 mmol/l Potassium 4.0 mmol/l Urea 4.2 mmol/l Creatinine 95 µmol/l ( ) ( ) ( ) (50 120) Should I be concerned? Point of reference needed 1 WEEK LATER Sodium 138 mmol/l Potassium 4.2 mmol/l Urea 4.8 mmol/l Creatinine 110 µmol/l Should I be concerned? Is this different Point of reference needed

5 An appreciation of analytical uncertainty in measurements. An understanding of biological variation in the measured parameter Points of reference (Context specific) An understanding of the contextual significance of the measured value.

6 Grasbeck & Saris 1969 Introduced the term reference value : The mode of generation of such values is known with respect to: - Selection of subjects Assessment of state of health Population characteristics, age, sex, Specimen collection and storage Analytical technique and performance characteristics Data handling techniques.

7 1. The Concept of Reference Values. 1987;25: The selection of Individuals for the Production of reference values. 1987;25: Preparation of individuals and collection of specimens for the production of reference intervals. 1988;26: Control of analytical variability in the production of reference values. 1991;29: Statistical treatment of collected reference limits. 1987;25: Presentation of observed values related to reference values. 1987;25: J Clin Chem Clin Biochem

8 Health associated reference values Subject based reference values Population based reference values Univariate Multivariate Time Specified FOCUS of IFCC Documents

9 Builds on IFCC work Specifically states, for the apriori approach, that the literature should be searched to identify known sources of biological variation: - Exclusion criteria Partitioning criteria Effects of biological variation on the analyte dictate criteria for subject selection and preparation.

10 1. Define the purpose for which they are to be used. 2. Only meaningful and transferable if defined for the population or individual in terms of: - Inclusion and exclusion criteria Intake of food & drugs Physiological and environmental conditions Specimen collection criteria Performance characteristics of the analytical method The statistical methods used for estimation of the limits Utility depends upon Knowledge of Biological Variation

11

12 Analytical variance. CV a Within Subject biological variance. CV i Between Subject biological variance. CV g s 2 Total = s 2 Analytical + s 2 Individual + s 2 Group CV Total = CV a + CV i + CV g

13 * * * * * * Subject 1 * * * * * * * * * * * * * * * * Subject 2 * * * * * * * * * * * * Subject 3 * * * * * * * * Analytical CVa Within Subject CVi Between Subject Variance

14 Potassium Alkaline Phosphatase Reference Range X Highly unusual result for the individual, but inside population reference interval LRL = Lower reference limit URL = Upper reference limit

15 Ratio of Within to Between subject variances. Index of Individuality (II) = [CV 2 a + CV i2 ] 1/2 /CV g CV i / CV g (close approximation if CV a <= CV i ) Population Ref Intervals: - Index <0.6 = Marked individuality Index >1.4 = Little individuality Implications for the use of population based ref intervals? Flagging significance of change?

16 Potassium Index of individuality = 0.86 Alkaline Phosphatase Index of individuality = 0.25 CV I = 4.8, CV G = 5.6 CV I = 6.4, CV G = 24.8 LRL = Lower reference limit URL = Upper reference limit

17

18

19 The majority of analytes demonstrate marked individuality Need for greater stratification of reference values. Issues with the data set

20 Urinary Free Noradrenaline. mol/mmol creatinine Index of individuality N MEAN CV a CV i CV g CV i / CV g [CV 2 a + CV i2 ] 1/2 /CV g MIXED SEX FEMALE MALE

21 Where individuality is marked the individual is the best point of reference. Difference > than combined analytical and biological variation: - RCV = 2 ½ * Z * (CV A 2 + CV I2 ) ½ The Z score determines the level of significance of the change: - e.g. 1 tailed 95% = % = 2.33

22 Sex Stratified Ref Range RCV Flag egfr > 60 in a 30 year old white female: Changing renal function?

23

24 Reference State: - The notion of reference state can be used to facilitate transferability of reference value data.

25 Qualification for Reference Status: years old Ideal body mass Fasted for 10 hours No medication Consuming <45 g of alcohol per day Smokes <12 cigarettes per day No apparent illness I m normal! I m a Clinical Chemist

26 Biological Rhythms (time) - Homeostasis Age Sex Stage of development Ethnicity State of well being Stimuli Socioeconomic impacts All provide criteria for stratification of reference values

27 Characterisation and understanding of biological variation enables a valid assessment of the significance of a laboratory result. Meta data are required to enable valid selection and application of data. This should include indices of biological variation

28 Only meaningful and transferable if defined for the population or individual in terms of: - Inclusion and exclusion criteria Intake of food & drugs Physiological and environmental conditions Specimen collection criteria Performance characteristics of the analytical method The statistical methods used for estimation of the limits

29

30

31 Less tight homeostatic control with age? Fraser 2001 Analyte Younger CVi Older CVi Sodium Potassium Chloride Urea Creatinine Calcium Cholesterol Proteins Albumin

32 Age 4 to 18 years 38 analytes: age partitioning required for 6 Creatinine, LDH, AST, ALP, Phosphate, Urate 14 showed marked differences from Westgard adult data

33

34 ACE Conc (U/L) Genotype n Assay 1* Assay 2* All D/D D/I I/I Genotype corrected reference intervals: SACE *mean +/ SD. D = deletion, I = Insertion.

35

36 3. State of health defined. WHO Defn: - a state of complete physical mental and social well being and not merely the absence of disease or infirmity Disease is a state of health. Conceptually different in different countries. The term Reference should be accompanied or preceded by a word qualifying the state of health. E.g diabetic, hospitalised diabetic, ambulatory diabetic, Healthy laboratory worker?

37 66 quantities 34 diseases with 45 references. For the majority of quantities studied CV I of same order as diseased. Disease specific RCVs may be necessary in some cases.

38 ISSUES Non-complex v complex molecules. Improved assay specificity. HbA1c PTH Creatinine

39 Study Year Subjects (M:F) State of Health Frequency of Sampling Number of Samples Method (6:4) Healthy 7 D IE (?) Diabetic 3-4 D 6 Endosmosis (?) Diabetic 1 M & 3 M 4 Affin Chrom (?) Diabetic 3 M & 12 M? HPLC IE (7:5) Healthy 15 D 10 HPLC IE (0:11) Healthy 7 D 5 HPLC IE (?) Diabetic 6 M 4-7 Imm Turbid (45:0) Diabetic 7 D 12 HPLC Affin (24:14) a Diabetic 1 Y 5 HPLC IE

40 H = Healthy D = Diabetic Study CV I CV G Analytical Goal Desirable TE A (%) Bias Target RCV N for Homeostatic Setting point 1 H D D 4.2 & & & & 10 4D H H <0.7 < ,5.4, 3.9 7D 8D 1.7 b ,2.7, ,16.7, ,6,3 9D

41 PTH Assays through time 1970 s C-Terminal RIA 1980 s Development of IRMA assays 1990 s Nichols institute Ruled the world Range of other intact assays with antibodies against a variety of epitopes 2004 Bioactive PTH Assays with n-terminal specific antibodies

42 If clearance of fragments is not identical in all patients and non diseased patients the apparent biological variation will vary and be assay specific.

43 Assay specificity is an important BV qualifier Historical data may not be always applicable.

44 Data in chronic stable disease often can be considered constant over time and geography Same order of magnitude in disease and health

45 Within Subject Variation (CV I,%) for Serum Sodium and Urea No. of Time Sex status Na + Urea subjects h m H h m H d H weeks m H weeks m H weeks F H weeks m H weeks m H months - H weeks - H d - RF weeks F HP weeks m DM Fraser 2001

46 Full Circle? Sodium 140 mmol/l Potassium 4.0 mmol/l Urea 4.2 mmol/l Creatinine 95 µmol/l 1 WEEK LATER Sodium 138 mmol/l Potassium 4.2 mmol/l Urea 4.8 mmol/l Creatinine 110 µmol/l Knowledge of biological variation within and between subjects provides a fundamental point of reference to enable interpretation of laboratory data.

47 Biological variation affects the clinical utility of reference values. An understanding of the nature of biological variation will:- enable production of relevant reference values enable effective application of clinical laboratory measurements We should consider additionally qualifying reference values by description of associated indices of biological variation. Biological variation data are reference data