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4 Sensitivity, Specificity, and Predictive Value
4.5 Random and Systematic Errors in Measurements
False positive results may mislead the clinician and lead to unnecessary
investigation and diagnostic tests as well as increased anxiety of the patient.
A false negative result is more dangerous than a false positive result
because diagnosis of a disease may be missed or delayed, which can
cause serious problems.
For a test, as clinical sensitivity increases, specificity decreases. For
calculating clinical sensitivity, specificity, and predictive value of a test,
the following formulas can be used:
TP 5 True positive (result correctly identifies a disease)
FP 5 False positive (result falsely identifies a disease)
TN 5 True negative (result correctly excludes a disease when the
disease is not present in an individual)
FN 5 False negative (result incorrectly excludes a disease when the
disease is present in an individual).
Therefore, when assay results are positive, results are a combination of TP
and FP, and when assay results are negative, results are combination of TN
and FN (Equations 4.6À4.8).
Sensitivity ðindividuals with disease who show positive test resultsÞ
TP 1 FN
Specificity (individuals without disease who show negative test results)
Positive predictive value 5
TN 1 FP
TP 1 FP
A positive predictive value is the proportion of individuals with disease who
showed a positive value compared to all individuals tested. Let us consider
an example where a particular analyte was measured in 100 normal individuals
and 100 individuals with disease. The following observations were made:
TP 5 95, FP 5 5, TN 5 95, and FN 5 5. Therefore, sensitivity 5 95/(95 1 5) 3
100 5 95%, and specificity 5 95/(95 1 5) 3 100 5 95%.
4.5 RANDOM AND SYSTEMATIC ERRORS
Random errors and systematic errors are important issues in the laboratory
quality control process. Random errors are unavoidable and occur due to
imprecision of an analytical method. On the other hand, systematic errors
have certain characteristics and are often due to errors in measurement using
Laboratory Statistics and Quality Control
a particular assay. Because random errors cannot be eliminated or controlled,
the goal of quality control in a clinical laboratory is to avoid or minimize
systematic errors. Usually recalibration of the assay is the first step taken by a
clinical laboratory technologist to correct systematic error, but more serious
problems such as instrument malfunction may also be responsible for systematic errors.
4.6 LABORATORY QUALITY CONTROL: INTERNAL
Good quality control is the heart of a good laboratory operation. Because the
value of an analyte in a patient’s specimen is unknown, clinical laboratory
professionals rely on producing accurate results using controls for an assay.
Controls can be purchased from a commercial source or can be made inhouse. A control is defined as a material that contains the analyte of interest
with a known concentration. It is important that the control material has a
similar matrix to serum or plasma. Different types of controls used in clinical
laboratories are listed below:
Assayed Control: The value of the analyte is predetermined. Most
commercially available controls have predetermined values of various
analytes. The target value must be verified before use.
Un-Assayed Control: The target value is not predetermined. This
control must be fully validated (run at least 20 times in a single run
and then run once a day for 20 consecutive days to establish a target
Homemade Control: If the assayed control material is not easily
commercially available (e.g. for an esoteric test), the control material may
be prepared by the laboratory staff by dissolving correctly weighed pure
material in an aqueous-based solvent or in serum or whole blood (for an
analyte not present in humans, e.g. a drug).
Commercially available control materials may be obtained as a ready-to-use
liquid control or as a lyophilized powder. If control material is available in
the form of lyophilized powder, it must be reconstituted prior to use by
strictly following the manufacturer’s recommended protocol. Control materials must be stored in a refrigerator following manufacturer’s recommendations and the expiration date of the control must be clearly visible so that an
expired control is not used by mistake. Usually low, medium, and high controls of an analyte are used to indicate analyte concentrations both in a normal physiological state and a disease state. At least two controls must be
used for each analyte (high and low controls). Control materials must be run
along with patient samples or at least once in each shift (a minimum of
three times in a 24 h period) depending on the assay.
4.6 Laboratory Quality Control: Internal and External
Quality control in the laboratory may be both internal and external. Internal
quality control is essential and results are plotted in a LeveyÀJennings chart
as discussed below. The most common example of external quality control is
analysis of CAP (College of American Pathologists) proficiency samples for
most tests offered by a clinical laboratory. Proficiency samples may not be
available for a few esoteric tests. CLIA 88 (Clinical Laboratory Improvement
Act) requires all clinical laboratories to register with the government and to
disclose all tests these laboratories offer. The test may be “waived tests” or
“Waived tests” are ones where laboratories can perform such tests as long
as they follow manufacturer protocol. Enrolling in an external
proficiency-testing program such as a CAP survey is not required for
“Non-waived tests” are moderately complex or complex tests.
Laboratories performing such tests are subjected to all CLIA regulations
and must be inspected by CLIA inspectors every two years or by
inspectors from non-government organizations such as CAP or Joint
Commission on Accreditation of Healthcare Organization (JCAHO). In
addition, a laboratory must participate in an external proficiency program
(most commonly CAP proficiency surveys) and must successfully pass
proficiency testing in order to operate legally. A laboratory must produce
correct results for four of five external proficiency specimens for each
analyte, and must have at least an 80% score for three consecutive
Since April 2003, clinical laboratories must perform method validation
for each new test, even if such test already has FDA approval.
Currently, most common external proficiency testing samples are offered by
CAP, and there are proficiency specimens for 580 analytes. The major
features of CAP external proficiency testing include:
CAP proficiency samples are mailed to participating laboratories three
times a year and there are at least five samples for each analyte during
CAP proficiency samples have matrix similar to patient specimens and
such specimens must be analyzed just like a regular patient specimen. For
example, a CAP specimen cannot be analyzed in duplicate or only on the
day shift; such practice to pass CAP proficiency testing is a violation of
established practice guidelines.
CAP proficiency testing results must be reported to CAP and later graded
or ungraded results must arrive at the laboratory for evaluation by
laboratory professionals. A laboratory director or designee must sign
results of a CAP survey and must act if the laboratory fails a survey.
Laboratory Statistics and Quality Control
CAP proficiency test results are graded based on performance of all
participating laboratories. There are various criteria for acceptability of a
result. Results must be within 6 2 SD of the peer group mean
(calculated by taking into account all values reported by participating
laboratories) or a fixed percentage of a target value (i.e. within 10% of
target value) or the result must be within a fixed deviation from the
target value (e.g. within 6 4 mol/L of the target value).
The best way to evaluate CAP proficiency testing results of an individual
clinical laboratory is to use the e-lab solution available from the CAP for
If CAP proficiency testing is not available, then the laboratory must
validate the test every six months by comparing values obtained by the
test with values obtained by a reference laboratory or another laboratory
offering the test (using split samples). Alternatively, if proficiency
samples are available from another source, for example, AACC (American
Association for Clinical Chemistry), passing such proficiency testing is
In addition to the CAP external proficiency-testing program, a laboratory
may participate in other proficiency testing programs. However, for
laboratory accreditation by CAP, it is required that the laboratory must
participate in a CAP proficiency survey, provided that the proficiency
specimen is available from the CAP.
There are a number of publications that indicate that participating in external
proficiency surveys such as offered by CAP is useful in improving the quality
of a clinical laboratory operation [1À3].
4.7 LEVEYÀJENNINGS CHART AND WESTGARD
In addition to participating in the CAP program, clinical laboratories must
run control specimens every shift, at least three times in a 24 h cycle. Also,
instruments must be calibrated as needed in order to maintain good laboratory practice. Calibration is needed for all assays that a clinical laboratory
offers. Calibration of immunoassays is discussed in Chapter 2. However,
other assays are calibrated using calibrators that are either commercially
available or homemade:
Calibrators are defined as materials that contain known amounts of the
analyte of interest. For a single assay, at least two calibrators are needed
for calibration, a zero calibrator (contains no analyte) and a high
calibrator containing the amount of the analyte that represents the upper
end of the analytical measurement range. However, five to six calibrators
4.7 LeveyÀJennings Chart and Westgard Rules
are commonly used for calibration. One calibrator must be a zero
calibrator and the highest calibrator must contain a concentration of the
analyte at the upper end of the analytical measurement range. Other
calibrators usually have concentrations in between the zero calibrator and
the highest calibrator, and represent normal values of the analyte as well
as values expected in a disease state (for drugs, values below therapeutic
range, between therapeutic ranges, and then toxic range).
Controls are materials that contain a known amount of the analyte. The
matrix of the control must be similar to the matrix of the patient’s
sample; for example, matrix of the control must resemble serum for
assays conducted in serum or plasma.
A LeveyÀJennings chart is commonly used for recording observed values of
controls during daily operation of a clinical laboratory. A LeveyÀJennings
chart is a graphical representation of all control values for an assay during an
extended period of laboratory operation. In this graphical representation,
values are plotted with respect to the calculated mean and standard deviation, and if all controls are within the mean and 6 2 SD, then all control
values are within acceptable limits and all runs during that period will have
acceptable performance (Figure 4.3). In this figure, all glucose low controls
were within acceptable limits for the entire month. The LeveyÀJennings chart
must be constructed for each control (low and high control or low, medium,
and high control) for each assay the laboratory offers. For example, if the laboratory runs two controls (low and high) for each test and offers 100 tests,
then there will be 100 3 2, or 200 LeveyÀJennings charts each month.
Usually a LeveyÀJennings chart is constructed for one control for one month.
The laboratory director or designee must review all LeveyÀJennings charts
each month and sign them for compliance with an accrediting agency.
+ 3 SD
+ 2 SD
+ 1 SD
– 1 SD
– 2 SD
– 3 SD
LeveyÀJennings chart with no violation.