Quality control in a Clinical Laboratory

Quality Control – Purpose and Importance

• Operational process control techniques to fulfill requirements for
quality and compliance.
• Quality control (QC) samples are tested identically to patient samples.
• QC ensures both precision and accuracy of patient samples results.
• The integrity of QC samples is important to both management of
overall quality as well as meeting requirements of proficiency testing.
• Addressing QC issues is critical to the identification of potential errors
with patient results, including reagent matrix effects as well as
calibration misalignment of testing function.
• Maintaining accurate and frequent checks of lab sample testing
through QC is vital to ensuring patient sample testing is done right.
Quality Control cont’d
• When QC works effectively, it is able to identify and correct errors in
the analytical processes of the laboratory before potentially incorrect
patient results are released.
• Failure of QC testing can result from methodological, technical,
proficiency testing material stability and random errors.
• By utilizing QC practices, a laboratory self-regulates its testing and
verifies that the results produced are accurate and precise.
• Clinical laboratories use management of documentation as well as
incorporation of a continual improvement process to well-organize
the overall quality control process.
Quality Control cont’d
• Other ways of managing QC include peer comparison & alternative
monthly review of QC trends.
• Clinical laboratories are frequently enrolled in proficiency testing (PT)
programs that are used to validate their testing protocols.
• These programs are utilised not only to validate laboratory testing but
to validate personnel training and procedures.
• Periodic review of QC results is a frequent tool for maintaining quality
control of patient samples.
• PT programs can also help laboratory personnel discover issues with
reagents even when controls and calibrators seem to be performing
well.
Levey-Jennings charts
• One of the most common tools used to track laboratory quality
control samples is the Levey-Jennings (L-J) chart.
• An L-J chart and the Westgard rules are frequently used to verify
trends, shifts, biases, or errors in quality controls.
• The Westgard rules observe the normal distribution expected and
identify standard deviations produced.
• Implementing Westgard rules within an L-J chart can identify violation
of the rules based on control limits established for the sample tested.
Levey-Jennings charts
• Many laboratories utilize L-J charts for 14- or 30-day reviews of QC
testing.
• While daily identification of QC deviations from normal ranges
ensures accuracy of sample testing, longer-term reviews are more
beneficial to diagnose trends and biases in tests which could be
missed on a daily basis.
• One concern with QC materials is discovering a “matrix-related bias
effect” which can skew normal results.
Levey-Jennings chart
Westgard Multirules
• Multirule QC uses a combination of decision criteria, or control rules,
to decide whether an analytical run is in-control or out-of-control.
• The well-known Westgard multirule QC procedure uses 5 different
control rules to judge the acceptability of an analytical run.
• By comparison, a single-rule QC procedure uses a single criterion or
single set of control limits, such as a Levey-Jennings chart with control
limits set as either the mean plus or minus 2 standard deviations (2s)
or the mean plus or minus 3s.
What are the Westgard rules?
• Westgard rules adopt a short hand notation to abbreviate different
decision criteria or control rules, e.g., 12s to indicate 1 control
measurement exceeding 2s control limits.
• 12srefers to the control rule that is commonly used with a Levey-
Jennings chart when the control limits are set as the mean plus/minus
2s (this rule is used as a warning rule to trigger careful inspection of
the control data by the following rejection rules).
• 13s refers to a control rule that is commonly used with a Levey-
Jennings chart when the control limits are set as the mean plus 3s
and the mean minus 3s. A run is rejected when a single control
measurement exceeds the mean plus 3s or the mean minus 3s
control limit.
Westgard rules con’t
• The "thumbnail" graphic next to a rule shows an example of control
results that violate that rule.
Westgard rule violation
• 22s - reject when 2 consecutive control measurements exceed the
same mean plus 2s or the same mean minus 2s control limit.
Westgard rule violation
• R4s - reject when 1 control measurement in a group exceeds the mean plus 2s and
another exceeds the mean minus 2s. This rule should only be interpreted within-
run, not between-run. The graphic below should really imply that points 5 and 6
are within the same run.
Westgard rule violation
• 41s - reject when 4 consecutive control measurements exceed the
same mean plus 1s or the same mean minus 1s control limit.
Westgard rule violation
• 10x - reject when 10 consecutive control measurements fall on one
side of the mean.
Westgard multirules
A diagram of Westgard multirules
Shift
• When six points in a row either are above or below the mean
Trend
• A trend is six points in an upward direction or six points in a row in a
downward direction
Qualitative Quality Control
• Qualitative quality control measure the presence or absence of a
substance, or evaluate cellular characteristics such as morphology.
• The results are not expressed numerically, but in descriptive or
qualitative terms such as “positive,” “negative,” “reactive,” “non-
reactive,” “normal,” or “abnormal.”
• Examples of qualitative examinations include microscopic
examinations for cell morphology or presence of parasitic organisms,
serologic procedures for presence or absence of antigens and
antibodies, some microbiological procedures, and some molecular
techniques.
Semi-quantitative tests
• Semi-quantitative tests are similar to qualitative examinations; testing
does not measure the precise quantity of a substance.
• The difference is that results of these tests are expressed as an
estimate of how much of a measured substance is present.
• Test results for semi-quantitative tests may be reported as “trace
amount”, “1+, 2+, or 3+”, or positive at 1:32 (titer or dilution).
• Examples of semi-quantitative examinations are serological
agglutination procedures.
QC - Conclusions
• Continuous monitoring of quality control testing and capture of
trends and biases are important to ensure accuracy of patient testing
results.
• The focus on trends and biases is a good identification of potential
changes in results that can affect accuracy of overall results.
• Management of matrix effects and calibration misalignment are
important aspects to observing shifting L-J charts and adjustments of
accuracy over time.
• Management of quality control can ensure accuracy and precision of
both quality and patient results.
Statistical tools in analytical method validation
• The objective of validation of an analytical method is to demonstrate that the
method is suitable for the intended use.
• The intent of method validation is to provide scientific evidence that the
analytical method is reliable and consistent before it can be used in routine
analysis of product.
• The primary parameters used for the interpretation of analytical method
validation results are the calculation of the mean, standard deviation, coefficient
of variation, confidence intervals, and regression analysis and the main tools are
the Fisher’s exact test (F-test), t-test, and regression and correlation analysis.
• These calculations are characteristically performed using statistical software
packages such as SPSS and Minitab.
• The goal of statistical analysis is to summarize a collection of data that provides
an understanding of the examined method characteristic.
Statistical tools in analytical method validation
Mean (or average)
X

where Xi are individual values and n is the number of individual data points

Standard deviation – measure of the spread of the values in the sample set

where Xi is individual value, X̄ is the sample mean, and n is the number of individual data points

Coefficient of variation (%CV)

where s is the standard deviation and x̄ is the sample mean

Statistical tools in analytical method validation
Confidence interval
• Confidence intervals indicate the reliability of an estimate.
• Confidence intervals provide limits around the sample mean to predict the range
of the true population mean.
• The prediction is usually based on probability of 95%.
• The confidence interval depends on the sample standard deviation and the
sample mean.
The hypothesis tests
Null hypothesis (H0) – no association
Alternative hypothesis (H1) – there is association
Test statistic (t) – is compared to the significance level
Statistical tools in analytical method validation
Correlation
• Correlation quantifies linear relationship between two sets of data but not the
agreement between them.
• The correlation coefficient (r) measures the strength of a relation between two
variables.
• The numerical value of r ranges from -1.0 to +1.0
• The closer the coefficients are to +1.0 or -1.0, the greater the strength of the
linear relationship is.
Statistical tools in analytical method validation
Linear regression
• Linear regression is used to evaluate a linear relationship between test results.
• A linear relationship is evaluated over the range of the analytical method.
• Linear regression evaluates the relationship between two variables by fitting a
linear equation/best fit line to observed data (Y = mx + b)
• Linear regression is commonly used to analyze method comparison data. Method
comparison can be considered a measure of accuracy.
• Coefficient of determination (R2) – main result of linear regression (ranges from 0
to 1). R2 of 0.98 or higher indicates good correlation between two methods.
• R2 and standard error are used to estimate random error.
• Standard error indicates the scatter of points about the regression line.
• Random error is a mistake in one test or test run that is independent of another
test or test run.
Statistical tools in analytical method validation
• Proportional error is an error that is dependent on the amount of
change in analyte concentration between the two methods.
• Constant error is a source of error that causes measurements to
deviate consistently from their ‘true’ value.
• In the absence of constant error, the regression line passes through the origin
and the Y intercept is 0.0.
• Systematic error is the sum of constant and proportional error and is
an indication of accuracy.
• Total error (TE) is random error plus systematic error.
Method validation performance characteristics
1. Specificity/selectivity
Specificity is a quantitative indication of the extent to which a
method can distinguish between the analyte of interest and
interfering substances on the basis of signals produced under actual
experimental conditions.
 Random interferences should be determined using representative
blank samples.
2. Sensitivity
Represent the smallest amount of substance in a sample that can
accurately be detected by an assay.
Method validation performance characteristics
3. Limit of detection
 Approaches for determining the detection limit include visual inspection & signal-to-noise ratio.
 If visual evaluation is used, the detection limit is determined by the analysis of samples with
known concentration of analyte and by establishing the minimum level at which the analyte can
be reliably detected.
 The signal-to-noise ratio is performed by comparing measured signals from samples with known
low concentrations of analyte with those of blank. Signal-to-noise ratio of 3:1 is acceptable.
4. Reportable and linear range
 A range of analyte concentrations over which the method may be applied.
 At the lower end of the concentration range the limiting factor is the value of the limit of
detection.
 At the upper end of the concentration range limitations will be imposed by various effects
depending on the detection mechanism.
 Within this reportable range there may exist a linear range, within which the detection response
will have a sufficiently linear relation to analyte concentration.
Method validation performance characteristics
5. Accuracy
Accuracy refers to closeness of agreement between the true value of the analyte
concentration and the mean result obtained by applying experimental procedure
to a large number of samples.
It is related to systematic error.
6. Precision
Repeatability measures the variation in measurements under the same operating
conditions. Repeatability is also termed within-run/intra-assay precision.
Reproducibility is the closeness of agreement between measurements performed
with the same method.
Both repeatability and the reproducibility are expressed in terms of mean,
standard deviation and coefficient of variation (%CV).
Method validation performance characteristics
7. Recovery experiment
 In recovery experiment, a known concentration of analyte is spiked
to a serum matrix.
 Then a test is run to measure the response of the spiked analyte
compared with a standard curve prepared with known
concentrations.
 Recovery experiments assess the degree of proportional error, which
is defined as 100 - % recovery.
 In other words recovery experiments assess accuracy of the method.
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