Assay Techniques in Clinical Chemistry

There are two fundamental assay techniques frequently used in clinical chemistry:

 End-point

 Rate

End-point assays:
Blank(s), standard(s)/calibrator(s) (if indicated in the procedure), controls and samples are
dispensed into the procedurally defined amount of reagent and incubated for a specified time
period under optimum reaction condition. At the end of the incubation time, when the reactions
have come to completion, the amount of product formed (for example, intensity of colour
developed or turbidity produced) is measured in terms of absorbance or other parameters. The
concentration of the serum analyte is then obtained from a pre-prepared calibration curve or
calculated using appropriate formula, eg, Lambert- Beer’s formula using a known standard or a
factor supplied by the manufacturer.

A unknown X Conc. calibrator/standard

Conc. unknown =
A calibrator/standard

Or

Conc. unknown = A unknown X Factor

Where : A standard/calibrator, control, sample = A standard, control, sample – A blank

Almost all substrates are determined by this assay technique – T. Proteins, cholesterol, albumin,
triglycerides, glucose (GOD-PAP method), urea (Bertholet method), bilurubin direct and total,
etc…
 N.B: Standards/calibrators are not run if a pre-prepared calibration curve exists or
a factor is supplied.
 If A calibrator/standard is found to be stable over time, the calibration (calculation)
factor obtained from a run, or better, a mean obtained from different runs can be
used as a permanent multiplication factor and obviate the need to include a
calibrator/standard in subsequent runs as far as quality control results are
acceptable.
Conc. calibrator/standard
Calibration (calculation) factor =
A calibrator / standard

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 Follow your test-specific SOP.
Rate assays:
In rate assays, repeated measurements are taken at procedurally defined time interval(s) over a
given total measurement time and the reaction rate or the mean reaction rate calculated will be
proportional to the concentration or volume activity of the serum analtye being investigated.

There are two types of rate assays:

 Initial rate or fixed-time kinetics

 Kinetic monitoring

Initial rate or fixed-time kinetics

In an initial rate or fixed-time kinetics, calibrator(s)/ standard(s) (if indicated in the procedure),
controls and samples are dispensed into the defined amount of reagent one at a time and
incubated for a specified time under optimum condition. An initial reading, A1, is taken at the
start of the reaction phase,T1. A second reading, A2, is then taken at the end of the time defined
for the reaction phase,T2. The A, A2-A1, observed for the calibrator(s) / standard(s), controls and
samples over the reaction phase are used to calculate the concentration of the analyte in the
controls and samples. Results may also be obtained from a pre-prepared calibration curve or by
using a multiplication factor supplied by the manufacturer.

A unknown X Conc. calibrator/standard

T2 - T1
Conc. unknown =
A calibrator/standard
T2 - T1
= A unknown X Conc. calibrator/standard
A calibrator/standard

Or

Conc. unknown = A unknown x Factor

Where :
A calibrator / standard, controls, unknown = A2 calibrator / standard, controls, unknown - A1 calibrator / standard, controls, unknown
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Urea (UV-method), creatinine (Jaffe reaction without deproteinization method) and glucose
determination by the Hexo-kinase procedure are some of the tests that employ initial rate assay
technique.

N.B:
 Standards/calibrators are not run if a pre-prepared calibration curve exists or a
factor is supplied.
 If Acalibrator/standard is found to be stable over time, the calibration
(calculation) factor

Conc. Calibrator/standard

A caliberator / standard

obtained from a run, or better, a mean obtained from different runs can be used as a permanent
multiplication factor and obviate the need to include a calibrator/standard in subsequent runs as
far as quality control results are acceptable.

 100 % T (zero absorbance) is generally set against dist. water or air.

 Never set against air if flow-through cell is used for measurements (it is never
dry!!!)

Follow your test-specific SOP.

Kinetic Monitoring

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In kinetic monitoring, calibrator(s)/standard(s) (if indicated in the procedure), controls and
samples are dispensed into the defined amount of reagent one at a time and incubated for a
specified time under optimum condition. Repeated (multiple) readings are taken at defined time
interval, also called read interval, over the procedurally defined total monitoring time or read
time. The following are then performed for each calibrator/standard, control and sample:

 A is calculated for each interval

 the As are added together to obtain total A over the monitoring time or read time
 the mean A/min is calculated by dividing the total A by the toal monitoring time or
read time in minutes. This mean reaction rate (mean A/min) relates proportionally to
the concentration or volume activity of the analyte being investigated.

Example: Suppose five absorbance readings, A0, A1, A2, A3 and A4 were recorded for a control
sample at 30 seconds’ read interval for two minutes.
 A1 = A1 - A0
 A2 = A2 - A1
 A3 = A3 – A2
 A4 = A4 -A3

Total A = A1 + A2 + A3 + A4

Mean A/min = A1 + A2 + A3 + A4

2 minutes

= 0.5 ( A1+ A2 + A3 + A4 )

min
Using the mean A/min, the control result may be calculated in one of the following ways:

 From a multi-point calibration curve previously prepared using

calibrators/standards

 Using a calibrator/standard of known concentration or volume activity

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 mean Acontrol /min X Conc. calibrator/standard
Conc. unknown =
mean A calibrator/standard/min

 Using a calculation factor provided by the manufacture or

derived from the following formula:

Where:
Factor (F) = Vt x 1000
 X Vs Vt = Total reaction volume = volume of reagent + Vs

Vs = Sample volume
 = molar extinction coefficient of the
substance monitored

Concentration / volume activity = mean A control / min X F

Remember that:

This is a standard assay technique in the determination of enzyme volume activities in controls
and samples.
The calculation factor attains either a positive or negative sign depending on whether an
increase in product or a decrease in reactant is monitored with time.
It is negative when a decrease in reactant is monitored and positive if product formation is
monitored.

Almost all enzymes (ALT, AST, ALP, LDH, CK, etc…) are determined by kinetic monitoring
assay technique.
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 N.B:

 100 % T (zero absorbance) is generally set against dist. water or air.

 Never set against air if flow-through cell is used for measurement (it is never dry!!!)

Follow your test-specific SOP.

Important terminologies in rate assays

Lag /delay time = the pre-measurement incubation time

Read/measurement time = the total length of time over which the reaction is monitored

Read/measurement interval = the time interval at which the intermediate readings are
taken and recorded for evaluation

Linearity tolerance (verification) value = the maximum allowable difference in

absorbance unit between As of the read intervals over the read time. This value may be
specified in terms of milli-absorbance (mA) or square multiple correlation coefficient, R 2,
which is a regression parameter for the monitored absorbance versus time curve. Both describe
the constancy or stability of the reaction rate over the read or measurement time. Best values
are  20mA or R 2 between 0.98 and 1.00. Values  20mA or  0.98 indicate a non-linear
reaction and gross variations between the As.
Samples with results accompanied by flags for non-linearity must be repeated after taking
appropriate corrective action for the cause  Refer to your test–specific SOP!!!.
N.B: Temperature and power fluctuations, dirty cuvettes, dirty and/or failing light source,
intermittent stray light, poor mixing, insufficient pre-incubation time, lipemic and hemolyzed
specimens are the main causes of non-linear reactions.

Substrate depletion value: This value specifies the absorbance of the reaction mixture at
which substrate concentration is too low to sustain reliable linear zero-order reaction kinetics.
This absorbance limit is usually reached as a result of very high concentration of analyte in the

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sample, but may also occur when expired or deteriorated or improperly prepared reagents are
used. An upper limit is specified for a reaction in which an increase in a product is monitored
and a lower limit is set for a reaction in which a decrease or consumption of a substrate or co-
substrate is followed-up.
Samples with results accompanied by flags for substrate depletion must be retested on
appropriately diluted sample and the result obtained multiplied by the dilution factor.
(Follow your test-specific SOP for the dilution procedure).

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