poor/average/excellent. The inverse of trueness,
which is called bias (or systemic error of measure-
ment), is a quantitative measure. It is the difference
between the true value of an analyte and the average
result value. Because it is useful to have a quantita-
tive measure of quality, bias is the measure that is
applied when appraising the trueness of a measure-
ment method.
Bias arises when the calibration process does not
reflect the test measurement process perfectly.
Causes of bias include matrix effects, calibrator
effects, and treatment effects (Strike 1996).
Matrix effects arise from the differences that
exist between the complex biological matrix found in
test samples and the artificial matrix of the calibra-
tors. Physical matrix effects are caused by physical
properties, such as sample viscosity, that result in
test samples being processed differently than calibra-
tors by the measurement instrument. Nonspecific
chemical matrix effects, called interference effects,
are caused by substances in the test sample that,
while not generating a signal themselves, affect the
magnitude of the signal generated by the analyte
being measured (Kroll and Elin 1994). Specific
chemical matrix effects are referred to as cross-
reaction effects. They are caused by substances in
the test sample that generate a signal identical to that
of the analyte of interest. Considerable effort is
devoted to the evaluation of cross-reactions during
the development of laboratory methods and various
techniques may be employed to improve the specific-
ity of the method by reducing or eliminating the
cross reacting substances. Separation of the analyte
from cross-reacting substances, discussed earlier as a
frequent step in the measurement process, is one
means of increasing method specificity. The selec-
tive adsorption of creatinine to fuller’s earth was
mentioned as an example. Some other separation
techniques are listed in Table 2.2. One of these
techniques, liquid chromatography, is even more
successful than fuller’s earth in specifically isolating
creatinine from substances that cross-react in the
Jaffé reaction. Method specificity can also be
improved in the analytical signal generation and
detection step of analyte measurement. One
approach taken at this step is to increase the selectiv-
ity of the signal generating reaction so that only the
specific analyte participates in the reaction. One
way to do this is to use analyte-specific enzymes to
catalyze a reaction that leads to the production of the
signal. A number of enzymatic methods are
available for the measurement of creatinine. In the
most popular method, creatinine is hydrolyzed to
creatine by the highly specific enzyme, creatinine
amidohydrolase. Creatine formation is coupled,
through a series of specific enzymatic reactions, to
the production of a light-absorbing species that
provides the analytical signal. The specificity inher-
ent in enzyme reactions can also be taken advantage
of when an enzyme is itself the analyte of interest.
In that case, a reagent that is a substrate of the
enzyme undergoes a catalytic conversion to a
product that is coupled to the production of the
analytical signal. For example, creatine kinase
concentrations are determined by measuring the rate
of formation of ATP and creatine from the substrates
ADP and creatine phosphate. Note that, in methods
of this sort, enzyme concentrations are measured and
reported in terms of enzymatic activity rather than
substance concentration. Another approach for
improving method specificity in the analytical signal
generation and detection step is to increase the selec-
tivity of signal detection so that the only the signal
generated by the analyte is detected. One way this is
done is by the so-called kinetic technique which
depends upon a differential rate of signal production
for the analyte and cross-reacting substances. In the
Jaffé reaction, the cross-reacting substances tend to
react with picrate slowly compared to creatinine so
the initial rate of Janovski complex formation is due
largely to creatinine. By measuring the initial rate,
the signal from creatinine can be segregated from the
signals arising from the cross-reacting substances.
Bias due to calibrator effects arises from differ-
ences between the analyte used in the calibrators and
the analyte as found in patients. One way this
happens is when a class of chemical species repre-
sents the analyte of interest but the calibration
material is based on a single species within the class.
The measurement of total protein in the urine is a
good example of this situation. Another cause of
calibration effects is the use of non-human or altered
human analyte in calibrators. Analytes used in
calibrators must be available in quantity and they
must be stable. It is often simply impossible to
procure from human sources adequate amounts of
trace analytes, such as hormones. It is similarly
impossible to preserve in unaltered form fragile
analytes such as blood cells.
Bias can also be caused by treatment effects.
These effects arise when test specimens and calibra-
tors are not treated in an identical fashion when
Laboratory Methods
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