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Biological Variation Dr WA Bartlett Biochemical Medicine Ninewells Hospital & Medical School DundeeScotland.

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Presentation on theme: "Biological Variation Dr WA Bartlett Biochemical Medicine Ninewells Hospital & Medical School DundeeScotland."— Presentation transcript:

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2 Biological Variation Dr WA Bartlett Biochemical Medicine Ninewells Hospital & Medical School DundeeScotland

3 Objectives Identification the nature of biological variation. Identification the nature of biological variation. Appreciation of the significance of biological variation in clinical measurements. Appreciation of the significance of biological variation in clinical measurements. Attain insight into the determination and application of indices of biological variation. Attain insight into the determination and application of indices of biological variation.

4 Identification the nature of biological variation. What is meant by the term biological variation in the context of clinical biochemistry? What is meant by the term biological variation in the context of clinical biochemistry? A component of the variance in biochemical measurements determined by the physiology of the subjects observed. A component of the variance in biochemical measurements determined by the physiology of the subjects observed.

5 Components of Variance in Clinical Chemistry Measurements Analytical variance. Analytical variance. Within Subject biological variance. Within Subject biological variance. Between Subject biological variance. Between Subject biological variance.

6 Biological Variation All clinical chemistry measurements change with time. All clinical chemistry measurements change with time. Knowledge of temporal changes useful in diagnosis and interpretation. Knowledge of temporal changes useful in diagnosis and interpretation. Rate of change may be useful in prognosis. Rate of change may be useful in prognosis. Understanding of the sources of biological variation in non-diseased subjects is fundamental to the development of reference data. Understanding of the sources of biological variation in non-diseased subjects is fundamental to the development of reference data.

7 Sources of Biological Variation Biological Rhythms (time) Biological Rhythms (time) Homeostasis Homeostasis Age Age Sex Sex Ethnicity Ethnicity Pathology Pathology Stimuli Stimuli

8 Practical significance of biological variation. What is the significance of this result? What is the significance of this result? Is the performance of the analytical method appropriate (imprecision, accuracy)? Is the performance of the analytical method appropriate (imprecision, accuracy)? When should I measure it again? When should I measure it again? Has this result changed significantly over time? Has this result changed significantly over time? Changes in variability be used as a tool? Changes in variability be used as a tool?

9 Models of Biological Variation Assume values represent random fluctuation around a homeostatic setting point. Assume values represent random fluctuation around a homeostatic setting point. More general model allows correlation between successive results. (Time series and non-decayed biological variation) More general model allows correlation between successive results. (Time series and non-decayed biological variation)

10 Quantifying Biological Variation How are you going to quantify biological variation? How are you going to quantify biological variation? You have to dissect out the components of variance: - You have to dissect out the components of variance: - total = Analytical + Individual + Group total = Analytical + Individual + Group

11 Quantifying Biological Variation Analytical = Individual = Group = Average variance of replicate assays within run analytical variance Average biological within subject variance. Average Variance around the homeostatic setting point Variance of true means among subjects. Variance in homeostatic setting points

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13 Quantifying Biological Variation How do you do the experiment? How do you do the experiment? SubjectsHow many? SubjectsHow many? Collect specimensNumber? Frequency? Collect specimensNumber? Frequency? Analyse specimens Minimise Analytical ? Analyse specimens Minimise Analytical ? Analyse dataOutliers? Statistics? Analyse dataOutliers? Statistics? Apply results of analysis. Apply results of analysis.

14 Quantifying Biological Variation Estimates of biological variation are similar regardless of: - Estimates of biological variation are similar regardless of: - Number of subjects Number of subjects Time scale of study (Short v Long?) Time scale of study (Short v Long?) Geography Geography A lot of information can be obtained from small studies. A lot of information can be obtained from small studies.

15 Within Subject Variation (CV I,%) for Serum Sodium and Urea No. ofTimeSex b statusNa + Urea subjects subjects hmH hmH dH weeksmH weeksmH weeksFH weeksmH weeksmH months-H weeks-H d-RF weeksFHP weeksmDM

16 Collection of Specimens. Conditions should minimise pre-analytical variables. Conditions should minimise pre-analytical variables. Healthy subjects. Healthy subjects. Usual life styles. Usual life styles. No drugs (alcohol, smoking?). No drugs (alcohol, smoking?). Phlebotomy by same person. Phlebotomy by same person. Same time of day at regular intervals. Same time of day at regular intervals. Set protocol for sample transport, processing & storage. Set protocol for sample transport, processing & storage.

17 Analysis of Specimens Need to minimise analytical imprecision. Need to minimise analytical imprecision. Ideal : - Ideal : - Single lots of reagents and calibrants. Single lots of reagents and calibrants. Single analyst and analytical system. Single analyst and analytical system. Single or very small number of batches. Single or very small number of batches.

18 Preferred Protocol: Cotlove et al Healthy subjects. Healthy subjects. Specimens taken at set time intervals. Specimens taken at set time intervals. Specimens processed & stored frozen. Specimens processed & stored frozen. When ALL specimens are available: - When ALL specimens are available: - Analysis of all samples in a single run. Simultaneous replicate analysis. Quality control to monitor drift

19 Preferred Protocol: Cotlove et al Advantage: - Advantage: - Minimisation of Analytical Disadvantages: - Disadvantages: - Limits the number of specimens and subjects that can be studied. Analyte must be stable on storage.

20 Other Protocols: Costongs et al Collection and storage as before. Collection and storage as before. Singleton assay of all samples in a single run. Singleton assay of all samples in a single run. Duplicate assay of QC or patient pool to estimate Analytical Duplicate assay of QC or patient pool to estimate Analytical

21 Other Protocols: Costongs et al Disadvantages: - Disadvantages: - True estimate of Analytical ? True estimate of Analytical ? Integrity of QC materials Integrity of QC materials Viral infections of pools Viral infections of pools Vial to vial variability in QC Vial to vial variability in QC

22 Other Protocols: Costongs/Moses et al Samples assayed once or in duplicate on the day of collection Samples assayed once or in duplicate on the day of collection Disadvantage: - Disadvantage: - individual confounded by between batch variance. individual confounded by between batch variance. Advantage: - Useful if analyte is unstable. Useful if analyte is unstable.

23 Analysis of Data 2 Stages 2 Stages – Identification of outliers – Nested analysis of variance

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25 Applications of BV Data Setting of analytical goals. Setting of analytical goals. Evaluating the significance of change in serial results. Evaluating the significance of change in serial results. Assessing the utility of reference intervals. Assessing the utility of reference intervals. Assessing number of specimens required to estimate homeostatic set points. Assessing number of specimens required to estimate homeostatic set points.

26 Applications of BV Data Assessment of reporting strategies. Assessment of reporting strategies. Selecting the best specimen. Selecting the best specimen. Comparing utility of available tests. Comparing utility of available tests.

27 Setting of analytical goals. Accepted analytical goal for imprecision: - Accepted analytical goal for imprecision: - CV Goal = ½ CV I therefore: - CV Analytical = CV Goal = ¼ of the Individual if achieved. (Harris. Am J Clin Pathol 1979:72;274)

28 Utility of Analytical Goals Assessment of methods and equipment. Assessment of methods and equipment. Should be addressed in early stages of method development. Should be addressed in early stages of method development. Index of Fiduciality: - Index of Fiduciality: - CV Analytical /CV Goal If <1 analytical goal met (Fraser Clin Chem 1988:34;995)

29 Evaluating the significance of change in serial results. Critical Difference or Reference Change value indicates the value by which 2 serial results must differ to be considered statistically significant: - Critical Difference or Reference Change value indicates the value by which 2 serial results must differ to be considered statistically significant: - CD = 2 ½ * Z * (CV A 2 + CV I 2 ) ½ Probabilty = 95% Z = 1.96 Probability = 99% Z = 2.58 Only valid if the variance of Individual is homogenous. Only valid if the variance of Individual is homogenous. (Costongs J Clin Chem Clin Biochem 1985;23:7-16)

30 Multipliers for (CV A 2 + CV I 2 ) ½ to Obtain Critical Difference at Different Levels of Probability Multiplier (2 ½ * Z) Probability of false alarm Probability99%95%90%80%70%60% 50%

31 Significance of Change? 63 year old patient: Cholesterol 1 = 6.60 mmol/L Cholesterol 2 = 5.82 mmol/L Cholesterol 2 = 5.82 mmol/L Significant change ? Cva = 1.6% CV I = 6.0% RCV = 2 ½ * Z * (CV A 2 + CV I 2 ) ½ 95%RCV = * 1.96 * (1.6 ½ ½ ) ½ = 17.2% 99%RCV = * 2.58 * (1.6 ½ ½ ) ½ = 22.6% Actual Change = ((6.60 – 5.82)/6.60)*100= 11.8%

32 Dispersion =Z* (SD 2 A + SD 2 I ) Dispersion of first result = result ± 1.96 SD : - 95% level 6.60 = 5.80 – % level 6.60 = 5.54 – 7.66 Dispersion of 2 result 95% level = 5.82 = 5.11 – % level = 5.82 = 4.89 – 6.75 Overlap: therefore neither significantly or highly significantly different Can use the formula to ascertain the probability that change is significant. Calculate Z using the ((( )/6.6)*100%) as RCV and look up in tables. 82% in this case.

33 USE of RCV Handbooks reports, 95% and 99% probabilities that change is significant. (> or >> * or **) Delta checking, exemption reporting. – 95% auto validate, 99% refer for clinical validation or renanalysis.

34 Index of Heterogeneity Measure of the heterogeneity of variance within the study population: - Measure of the heterogeneity of variance within the study population: - ratio of the observed CV of the set of subjects variances (SD A+I 2 ) to the theoretical CV ( / 2/n-1) for the set. The ratio should =1 (1SD = 1/ /2n ) The ratio should =1 (1SD = 1/ /2n ) Large ratio = more heterogeneity. Large ratio = more heterogeneity. (Costongs J Clin Chem Clin Biochem 1985;23:7-16) (Costongs J Clin Chem Clin Biochem 1985;23:7-16)

35 Assessing the utility of reference intervals. Utility of population based reference data? Utility of population based reference data? Ratio of Within to Between subject variances. Ratio of Within to Between subject variances. Index of Individuality = CV I / CV G Population Ref Intervals: - Index <0.6 = Limited in Value Index <0.6 = Limited in Value Index >1.4 = Applicable

36 Biological Variation &Utility of Reference Intervals

37 Number of specimens required to estimate homeostatic set points. n = ( Z. CV A+ I /D) n = ( Z. CV A+ I /D) where: - where: - Z = number of Standard deviates for a stated probablity (e.g for 95%). Z = number of Standard deviates for a stated probablity (e.g for 95%). D = desired % closeness homeostatic set point. D = desired % closeness homeostatic set point.

38 Number of specimens required to estimate homeostatic set points: - Cholesterol testing How many samples (n) required to estimate set point within ±5% given: - CV I = 4.9%CV A = 3% (Recommended) Substitute equation: - n = ( Z. CV A+ I /D) n = ( Z. CV A+ I /D) n = [1.96·( ) ½ /5] 2 = 5.07 n = [1.96·( ) ½ /5] 2 = 5.07

39 RCV at 95% and Number. of Specimens Required to Assess the Homeostatic Set Point at Different Levels of Imprecision CV A CV I RCV a Number of (%)(%) (%) specimens b a RCV (p <0.05) = 2.77 (CV A 2 + CV I 2 ) ½, assuming no statistical evidence of heterogenity b Number = mean result is within 5%of homeostatic set point x (CV A 2 + CV I 2 ) ½ /25.

40 Assessment of reporting strategies Results may be reported in different formats Results may be reported in different formats e.g. 24h Urinary creatinine output: - CV I for concentration = 23.8% CV I for output per collection = 13.0% CD for concentration = 66.0% CD for output = 36.2%

41 Selecting best Specimen. e.g early morning urines for albumin versus 24h collections. e.g early morning urines for albumin versus 24h collections. Random hormone measurements versus timed measurements. Random hormone measurements versus timed measurements.

42 Comparing Available Tests Creatinine v Creatinine Clearance Creatinine v Creatinine Clearance FT4 v TSH in replacement situations FT4 v TSH in replacement situations FT4 v Total T4 FT4 v Total T4

43 Reference Intervals Dr WA Bartlett Birmingham Heartlands & Solihull NHS Trust (Teaching)

44 WHO Definition of Health "a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity" "a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity"

45 Grasbeck 1981: - "Health is characterized by a minimum of subjective feelings and objective signs of disease, assessed in relation to the social situation of the subject and the purpose of the medical activity, and is in the absolute sense an unattainable ideal state "Health is characterized by a minimum of subjective feelings and objective signs of disease, assessed in relation to the social situation of the subject and the purpose of the medical activity, and is in the absolute sense an unattainable ideal state Thus, health is a goal-oriented concept more than a "state" mentioned in the WHO definition

46 IFCC Definition of Health health is said to be a relative and not an absolute state, it being conceptually different in different countries, in the same country at different times and in the same individual at different ages health is said to be a relative and not an absolute state, it being conceptually different in different countries, in the same country at different times and in the same individual at different ages


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