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What types of data fit a Gaussian distribution?S#0H Like the ski resort full of girls hunting for husbands and husbands hunting for girls, the situation is not as symmetrical as it might seem. Alan Lindsay Mackay (1926- )IAre these Gaussian?{Human height Outside temperature Raindrop size Blood glucose concentration Serum CK activity QC results Proficiency results||JThe Gaussian DistributionWhat is the Gaussian distribution? What types of data fit a Gaussian distribution? What is the advantage of using a Gaussian distribution?S8K!Gaussian probability distributionL)What are the odds of an observation . . .more than 1 s from the mean (+/-) more than 2 s greater than the mean more than 3 s from the mean~b  M"Some useful Gaussian probabilitiesNExampleO&[On the Gaussian curve]  Experimentalists think that it is a mathematical theorem while the mathematicians believe it to be an experimental fact. {Gabriel Lippman (1845-1921) P Distributions%Definition Examples Binomial Gaussian&QU"Life is good for only two things, discovering mathematics and teaching mathematics" VTSimon Poisson (1781-1840)  RThe Poisson DistributionThe Poisson distribution predicts the frequency of r events occurring randomly in time, when the expected frequency is l2y3BS6Examples of events described by a Poisson distributionLightning Accidents Laboratory? U2A very useful property of the Poisson distributionVUsing the Poisson distribution^How many counts must be collected in an RIA in order to ensure an analytical CV of 5% or less?__WAnswerX Distributions-Definition Examples Binomial Gaussian Poisson&Y8The Student s t DistributionWhen a small sample is selected from a large population, we sometimes have to make certain assumptions in order to apply statistical methods\Questions about our sampleIs the mean of our sample, x bar, the same as the mean of the population, m? Is the standard deviation of our sample, s, the same as the standard deviation for the population, s? Unless we can answer both of these questions affirmatively, we don t know whether our sample has the same distribution as the population from which it was drawn.dUJ+9] Recall that the Gaussian distribution is defined by the probability function: Note that the exponential factor contains both m and s, both population parameters. The factor is often simplified by making the substitution:8Z^ The variable z in the equation: is distributed according to a unit gaussian, since it has a mean of zero and a standard deviation of 18 2<_!Gaussian probability distribution` But if we use the sample mean and standard deviation instead, we get: and we ve defined a new quantity, t, which is not distributed according to the unit Gaussian. It is distributed according to the Student s t distribution.LZk QcdImportant features of the Student s t distributionUse of the t statistic assumes that the parent distribution is Gaussian The degree to which the t distribution approximates a gaussian distribution depends on N (the degrees of freedom) As N gets larger (above 30 or so), the differences between t and z become negligible dpApplication of Student s t distribution to a sample meanThe Student s t statistic can also be used to analyze differences between the sample mean and the population mean: vvehComparison of Student s t and Gaussian distributionsqNote that, for a sufficiently large N (>30), t can be replaced with z, and a Gaussian distribution can be assumed8r--fExerciseThe mean age of the 20 participants in one workshop is 27 years, with a standard deviation of 4 years. Next door, another workshop has 16 participants with a mean age of 29 years and standard deviation of 6 years. Is the second workshop attracting older technologists?"Z8iPreliminary analysisrIs the population Gaussian? Can we use a Gaussian distribution for our sample? What statistic should we calculate?ssjSolution3First, calculate the t statistic for the two means:&4kSolution, cont.'Next, determine the degrees of freedom:((lStatistical Tables m ConclusionSince 1.16 is less than 1.64 (the t value corresponding to 90% confidence limit), the difference between the mean ages for the participants in the two workshops is not significant nThe Paired t TestSuppose we are comparing two sets of data in which each value in one set has a corresponding value in the other. Instead of calculating the difference between the means of the two sets, we can calculate the mean difference between data pairs.o 'Instead of: we use: to calculate t:((pAdvantage of the Paired toIf the type of data permit paired analysis, the paired t test is much more sensitive than the unpaired t. Why?0kkqApplications of the Paired t*Method correlation Comparison of therapies++r DistributionsrDefinition Examples Binomial Gaussian Poisson Student s t&&&s@The c2 (Chi-square) Distribution:!UThere is a general formula that relates actual measurements to their predicted valuesVVt@The c2 (Chi-square) Distribution8!A special (and very useful) application of the c2 distribution is to frequency data@T/ #uExerciseIn your hospital, you have had 83 cases of iatrogenic strep infection in your last 725 patients. St. Elsewhere, across town, reports 35 cases of strep in their last 416 patients. Do you need to review your infection control policies? 7vAnalysisIf your infection control policy is roughly as effective as St. Elsewhere s, we would expect that the rates of strep infection for the two hospitals would be similar. The expected frequency, then would be the average&&wCalculating c2& VFirst, calculate the expected frequencies at your hospital (f1) and St. Elsewhere (f2)LW<xCalculating c2& LNext, we sum the squared differences between actual and expected frequenciesMMyDegrees of freedom2In general, when comparing k sample proportions, the degrees of freedom for c2 analysis are k - 1. Hence, for our problem, there is 1 degree of freedom.T0=z ConclusionA table of c2 values lists 3.841 as the c2 corresponding to a probability of 0.05. So the variation (c2 = 2.34) between strep infection rates at the two hospitals is within statistically-predicted limits, and therefore is not significant. )  { DistributionsxDefinition Examples Binomial Gaussian Poisson Student s t c2P)&H}The F distribution$The F distribution predicts the expected differences between the variances of two samples This distribution has also been called Snedecor s F distribution, Fisher distribution, and variance ratio distributionX< 7P F~The F distribution$fThe F statistic is simply the ratio of two variances (by convention, the larger V is the numerator)&gb"Applications of the F distribution$#There are several ways the F distribution can be used. Applications of the F statistic are part of a more general type of statistical analysis called analysis of variance (ANOVA). We ll see more about ANOVA later.J0J-ExampleYou re asked to do a  quick and dirty correlation between three whole blood glucose analyzers. You prick your finger and measure your blood glucose four times on each of the analyzers. Are the results equivalent? Data AnalysisThe mean glucose concentrations for the three analyzers are 70, 85, and 76. If the three analyzers are equivalent, then we can assume that all of the results are drawn from a overall population with mean m and variance s2.L@Analysis, cont.fApproximate m by calculating the mean of the means:&4 'Analysis, cont.$Calculate the variance of the means:%%Analysis, cont.HBut what we really want is the variance of the population. Recall that:IIAnalysis, cont.\Since we just calculated we can solve for s:/,Analysis, cont.So we now have an estimate of the population variance, which we d like to compare to the real variance to see whether they differ. But what is the real variance? We don t know, but we can calculate the variance based on our individual measurements.*Z WAnalysis, cont.If all the data were drawn from a larger population, we can assume that the variances are the same, and we can simply average the variances for the three data sets.Analysis, cont.Now calculate the F statistic:&  ConclusionA table of F values indicates that 4.26 is the limit for the F statistic at a 95% confidence level (when the appropriate degrees of freedom are selected). Our value of 10.6 exceeds that, so we conclude that there is significant variation between the analyzers.8 0 Distributions|Definition Examples Binomial Gaussian Poisson Student s t c2 FT+&!Unknown or irregular distribution Transform  Log transform!Unknown or irregular distribution Transform Non-parametric methods! Non-parametric methodsNon-parametric methods make no assumptions about the distribution of the data There are non-parametric methods for characterizing data, as well as for comparing data sets These methods are also called distribution-free, robust, or sometimes non-metric testsLZ  Application to Reference RangesThe concentrations of most clinical analytes are not usually distributed in a Gaussian manner. Why? How do we determine the reference range (limits of expected values) for these analytes?,#  Application to Reference RangesReference ranges for normal, healthy populations are customarily defined as the  central 95% . An entirely non-parametric way of expressing this is to eliminate the upper and lower 2.5% of data, and use the remaining upper and lower values to define the range. NCCLS recommends 120 values, dropping the two highest and two lowest.KKApplication to Reference RangesWhat happens when we want to compare one reference range with another? This is precisely what CLIA  88 requires us to do. How do we do this? | Everything should be made as simple as possible, but not simpler. DDAlbert Einstein Solution #1: Simple comparisonFSuppose we just do a small internal reference range study, and compare our results to the manufacturer s range. How do we compare them? Is this a valid approach?4qSNCCLS recommendationsInspection Method: Verify reference populations are equivalent Limited Validation: Collect 20 reference specimens No more than 2 exceed range Repeat if failed Extended Validation: Collect 60 reference specimens; compare ranges. r-F, -.Solution #2: Mann-Whitney*Rank normal values (x1,x2,x3...xn) and the reference population (y1,y2,y3...yn): x1, y1, x2, x3, y2, y3 ... xn, yn Count the number of y values that follow each x, and call the sum Ux. Calculate Uy also. *Also called the U test, rank sum test, or Wilcoxen s test.RZ#ZZ             B        +A 2 Mann-Whitney, cont.It should be obvious that: Ux + Uy = NxNy If the two distributions are the same, then: Ux = Uy = 1/2NxNy Large differences between Ux and Uy indicate that the distributions are not equivalent,-W    .      4/%4p  Obvious is the most dangerous word in mathematics. 97Eric Temple Bell (1883-1960)Solution #3: Run test In the run test, order the values in the two distributions as before: x1, y1, x2, x3, y2, y3 ... xn, yn Add up the number of runs (consecutive values from the same distribution). If the two data sets are randomly selected from one population, there will be few runs.PG#8    B    ,b$Solution #4: The Monte Carlo methodSometimes, when we don t know anything about a distribution, the best thing to do is independently test its characteristics.}}The Monte Carlo methodThe Monte Carlo methodThe Monte Carlo methodWith the Monte Carlo method, we have simulated the test we wish to apply--that is, we have randomly selected samples from the parent distribution, and determined whether our in-house data are in agreement with the randomly-selected samples.Analysis of paired dataFor certain types of laboratory studies, the data we gather is paired We typically want to know how closely the paired data agree We need quantitative measures of the extent to which the data agree or disagree Examples?J?7[Examples of paired dataMMethod correlation data Pharmacodynamic effects Risk analysis Pathophysiology$ Correlation!Linear regression (least squares)Linear regression analysis generates an equation for a straight line y = mx + b where m is the slope of the line and b is the value of y when x = 0 (the y-intercept). The calculated equation minimizes the differences between actual y values and the linear regression line.E E    (H Correlation Covariance/Do x and y values vary in concert, or randomly?80& oWhat if y increases when x increases? What if y decreases when x increases? What if y and x vary independently?p    CovarianceIt is clear that the greater the covariance, the stronger the relationship between x and y. But . . . what about units? e.g., if you measure glucose in mg/dL, and I measure it in mmol/L, who s likely to have the highest covariance?ZS /,1The Correlation CoefficientThe Correlation CoefficientThe correlation coefficient is a unitless quantity that roughly indicates the degree to which x and y vary in the same direction. r is useful for detecting relationships between parameters, but it is not a very sensitive measure of the spread.^i  Correlation CorrelationStandard Error of the EstimateThe linear regression equation gives us a way to calculate an  estimated y for any given x value, given the symbol w (y-hat):LJ  Standard Error of the EstimateNow what we are interested in is the average difference between the measured y and its estimate, w ::eM  Correlation Correlation Standard Error of the EstimateIf we assume that the errors in the y measurements are Gaussian (is that a safe assumption?), then the standard error of the estimate gives us the boundaries within which 67% of the y values will fall. 2sy/x defines the 95% boundaries..tZ$[  Limitations of linear regressiongAssumes no error in x measurement Assumes that variance in y is constant throughout concentration rangeJh&, Alternative approachesWeighted linear regression analysis can compensate for non-constant variance among y measurements Deming regression analysis takes into account variance in the x measurements Weighted Deming regression analysis allows for bothK8%Evaluating method performance Precision Method PrecisionWithin-run: 10 or 20 replicates What types of errors does within-run precision reflect? Day-to-day: NCCLS recommends evaluation over 20 days What types of errors does day-to-day precision reflect?T!868!868Evaluating method performancePrecision Sensitivity Method Sensitivity The analytical sensitivity of a method refers to the lowest concentration of analyte that can be reliably detected. The most common definition of sensitivity is the analyte concentration that will result in a signal two or three standard deviations above background.0  Other measures of sensitivityLimit of Detection (LOD) is sometimes defined as the concentration producing an S/N > 3. In drug testing, LOD is customarily defined as the lowest concentration that meets all identification criteria. Limit of Quantitation (LOQ) is sometimes defined as the concentration producing an S/N >5. In drug testing, LOQ is customarily defined as the lowest concentration that can be measured within 20%.Yp[jYp[d&QuestionAt an S/N ratio of 5, what is the minimum CV of the measurement? If the S/N is 5, 20% of the measured signal is noise, which is random. Therefore, the CV must be at least 20%.  BqEvaluating method performancePrecision Sensitivity Linearity  Method LinearityA linear relationship between concentration and signal is not absolutely necessary, but it is highly desirable. Why? CLIA  88 requires that the linearity of analytical methods is verified on a periodic basis.Ways to evaluate linearityVisual/linear regressionOutliersWe can eliminate any point that differs from the next highest value by more than 0.765 (p=0.05) times the spread between the highest and lowest values (Dixon test). Example: 4, 5, 6, 13 (13 - 4) x 0.765 = 6.89(&&Limitation of linear regression methodIf the analytical method has a high variance (CV), it is likely that small deviations from linearity will not be detected due to the high standard error of the estimateWays to evaluate linearity-Visual/linear regression Quadratic regression.Quadratic regressioneRecall that, for linear data, the relationship between x and y can be expressed as y = f(x) = a + bxT7bQuadratic regressionA curve is described by the quadratic equation: y = f(x) = a + bx + cx2 which is identical to the linear equation except for the addition of the cx2 term.1T   I?[ Quadratic regressionIt should be clear that the smaller the x2 coefficient, c, the closer the data are to linear (since the equation reduces to the linear form when c approaches 0). What is the drawback to this approach?`(X'!Ways to evaluate linearityBVisual/linear regression Quadratic regression Lack-of-fit analysisC.$Lack-of-fit analysis There are two components of the variation from the regression line Intrinsic variability of the method Variability due to deviations from linearity The problem is to distinguish between these two sources of variability What statistical test do you think is appropriate?BCQzCQz%&Lack-of-fit analysisThe ANOVA technique requires that method variance is constant at all concentrations. Cochran s test is used to test whether this is the case.'Lack-of-fit method calculations2Total sum of the squares: the variance calculated from all of the y values Linear regression sum of the squares: the variance of y values from the regression line Residual sum of the squares: difference between TSS and LSS Lack of fit sum of the squares: the RSS minus the pure error (sum of variances)3Z*%!!2(Lack-of-fit analysis4The LOF is compared to the pure error to give the  G statistic (which is actually F) If the LOF is small compared to the pure error, G is small and the method is linear If the LOF is large compared to the pure error, G will be large, indicating significant deviation from linearity~32S@)Significance limits for GA90% confidence = 2.49 95% confidence = 3.29 99% confidence = 5.42* If your experiment needs statistics, you ought to have done a better experiment. SSErnest Rutherford (1871-1937),3Evaluating Clinical Performance of laboratory testsThe clinical performance of a laboratory test defines how well it predicts disease The sensitivity of a test indicates the likelihood that it will be positive when disease is presentJ? U-Clinical SensitivityIf TP as the number of  true positives , and FP is the number of  false positives , the sensitivity is defined as:Js() .ExampleOf 25 admitted cocaine abusers, 23 tested positive for urinary benzoylecgonine and 2 tested negative. What is the sensitivity of the urine screen? g-/3Evaluating Clinical Performance of laboratory testsThe clinical performance of a laboratory test defines how well it predicts disease The sensitivity of a test indicates the likelihood that it will be positive when disease is present The specificity of a test indicates the likelihood that it will be negative when disease is absentd? Y T0Clinical SpecificityIf TN is the number of  true negative results, and FP is the number of falsely positive results, the specificity is defined as:J/0 1ExamplenWhat would you guess is the specificity of any particular clinical laboratory test? (Choose any one you want)oo2AnswerSince reference ranges are customarily set to include the central 95% of values in healthy subjects, we expect 5% of values from healthy people to be  abnormal --this is the false positive rate. Hence, the specificity of most clinical tests is no better than 95%. E3Sensitivity vs. Specificity2Sensitivity and specificity are inversely related.3345Sensitivity vs. SpecificitySensitivity and specificity are inversely related. How do we determine the best compromise between sensitivity and specificity?3M6!Receiver Operating Characteristic73Evaluating Clinical Performance of laboratory tests3The sensitivity of a test indicates the likelihood that it will be positive when disease is present The specificity of a test indicates the likelihood that it will be negative when disease is absent The predictive value of a test indicates the probability that the test result correctly classifies a patientl4 Y TY8Predictive ValueThe predictive value of a clinical laboratory test takes into account the prevalence of a certain disease, to quantify the probability that a positive test is associated with the disease in a randomly-selected individual, or alternatively, that a negative test is associated with health.  9 IllustrationSuppose you have invented a new screening test for Addison disease. The test correctly identified 98 of 100 patients with confirmed Addison disease (What is the sensitivity?) The test was positive in only 2 of 1000 patients with no evidence of Addison disease (What is the specificity?):X:Test performanceThe sensitivity is 98.0% The specificity is 99.8% But Addison disease is a rare disorder--incidence = 1:10,000 What happens if we screen 1 million people?o,;AnalysisIn 1 million people, there will be 100 cases of Addison disease. Our test will identify 98 of these cases (TP) Of the 999,900 non-Addison subjects, the test will be positive in 0.2%, or about 2,000 (FP).JkZ<%Predictive value of the positive testGThe predictive value is the % of all positives that are true positives:HH=)What about the negative predictive value?9TN = 999,900 - 2000 = 997,900 FN = 100 * 0.002 = 0 (or 1).:>Summary of predictive valuegPredictive value describes the usefulness of a clinical laboratory test in the real world. Or does it?.\ \ ?Lessons about predictive valueEven when you have a very good test, it is generally not cost effective to screen for diseases which have low incidence in the general population. Exception? The higher the clinical suspicion, the better the predictive value of the test. Why?F /@ EfficiencyWe can combine the PV+ and PV- to give a quantity called the efficiency: The efficiency is the percentage of all patients that are classified correctly by the test result.` hA Efficiency of our Addison screenBd To call in the statistician after the experiment is done may be no more than asking him to perform a postmortem examination: he may be able to say what the experiment died of. "Ronald Aylmer Fisher (1890 - 1962)##E,Application of Statistics to Quality Control5We expect quality control to fit a Gaussian distribution We can use Gaussian statistics to predict the variability in quality control values What sort of tolerance will we allow for variation in quality control values? 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