read after a fixed time interval. Hence, for running one assay twice, double the amount of activation buffer and sample will have to be used.

 

 the assay system will operate accurately for the

said analyte and a concentration value of the sample can

be interpolated.

The interval between the signal generated by the lowest

calibrator to the signal generated by the highest calibrator,

which gives proportionate and measurable and a linear

signal, is referred to as the measuring range of the assay

system.

Security Range

The critical point (Cx in Fig. 23.13), in the antigen excess

zone of the dose-response curve corresponds to the

maximum concentration value of analyte, which gives a

signal value higher than the signal value of the calibrator

of highest concentration, and just before the value (B1 in

FIG. 23.10: Signal development as a function of time. Figure

illustrating ‘immediate mixed blanking’ using IgA as an example

FIG. 23.11: Signal development as a function of time. Figure

illustrating ‘immediate mixed blanking’ using IgG as an example

FIG. 23.12A: Standard curves for IgA obtained with ‘real sample

blanking’ and ‘immediate mixed blanking’

Fig. 23.13) at which erroneous interpolation begins. The

interval between the signal of highest standard and the

signal of the critical concentration can be referred to as the

security range of the assay system for the analyte.

Reagent Optimization

The risk of obtaining signals in the antigen excess zone

are more relevant in analytes like C-reactive protein or

immunoglobulins such as IgG where the concentration

between normal and pathological sample can differ

by manifolds. It is necessary to make sure that the

concentration values lying in the antigen excess zone

(critical point) are beyond the concentrations which can

712 Concise Book of Medical Laboratory Technology: Methods and Interpretations be expected to occur in clinical samples during routine

analysis.

The relation between the security range and measuring

range is very important in optimizing assay systems.

As mentioned earlier the shape of the dose-response

curve depends on the ratio between the antigen and

the antibody. At a constant antibody concentration, an

increase in the measuring range will result in a narrower

security range, leading to antigen excess at a lower antigen

concentration (Fig. 23.14).

The implications of increasing the antibody reagent

concentrations can be practically demonstrated using IgG as

an analyte and antihuman IgG as a reagent. The measuring

range and the security range can be expanded by increasing

the antibody concentration. However, this expansion can

only be done to a point where it is still possible to have the

desired sensitivity for lower analyte concentration.

Figure 23.15, shows the effect of increasing the antibody

concentration on the security ranges while keeping

constant, the measuring range of the dose-response curve.

When the volume of an antibody solution of concentration

‘X’ used is 50 µL, the security range obtained is around 5000

mg/dL. As the volume of the same antibody concentration is

increased to 75 µL, the security range increases to >10,000 mg/

dL. With a further increase in volume of the same antibody

concentration to 100 µL, the security range shifts to >15,000

mg/dL. But this increase in measuring range is possible till a

certain limit of increasing volume of antibody solution. If the

volume of antibody solution is increased further, there will

be a decrease in the absorbance at the lowest concentration

of the measuring range due to antibody excess, resulting in

compromising with assay sensitivity.

By adjusting the sample dose and the antibody

concentration, a measuring range 20 to 25 times the lowest

calibrator value can be possibly optimized, with a security

range still giving a warning up to the pathophysiological

concentration. A wide measuring range combined with a

wide security range offer the advantage of a few reruns and

maximum security against antigen excess problems.

Standard Curve

Once the dose-response curve for a reagent has been

optimized, a standard curve can be obtained by using

a number of dilutions of the calibrator (preferably 5–6)

covering the optimal measuring range. The lowest

calibrator should be chosen to give a signal significantly

higher than the background noise. The highest calibrator

should be selected to allow measurements for a reasonably wide range of analyte concentrations, and still leaving

space for a fair security range (Fig. 23.16).

A curve is fitted to the signals obtained for calibrator

dilutions and can be stored in the memory of the

instrument. Different curve fitting programs can be made

available in instrument software.

Many of the instruments are equipped with a facility

to give a ‘warning’ that indicate reruns of the test with

FIG. 23.12B: Standard curves for IgG obtained with ‘real sample

blanking’ and ‘immediate mixed blanking’

FIG. 23.13: Dose-response curve Figure illustrating measuring range

(X-Y), critical concentration (Z), standard curve (A1-A6), erroneous

interpolation (where Abs. val.1.0 corresponds to A6 and B1, Abs. val.

0.8 corresponds to A5 and B2 and Abs val. 0.6 corresponds to A4 and

B3), security range (Y-Z)

Diagnostic Immunology 713

dilution of the sample with high concentration values of

analyte. This warning is given as long as the sample signal

is higher than the signal of the highest calibrator.

The validity of the stored standard curve should be

checked with known controls at regular time intervals.

Instrumentation for Turbidimetry

The development of automated instruments for the

clinical laboratory began in the 1950s at the same time

as the demand for test such as IgA, CRP, HBsAg escalated

dramatically. One of the benefits of automation is a

reduction in the variability of results and errors of analysis

by eliminating tasks that are repetitive and monotonous

for a human and that can lead to boredom or inattention.

The significant improvement in quality of laboratory tests

in recent years owes much to the combination of welldesigned instrumentation with good analytical methods.

The photometric requirements of turbidimetric analysis

are no different from those of photometric biochemistry

analysis. However, the photometer used must be provided

with means to maintain the contents of the cuvette at

a constant temperature during the reaction along with

compatible software to run the various steps of the reagent

and reagent kinetics appropriately.

The chemistry analyzers (spectrophotometers)

available utilize either or both of the two modes of

measuring absorbance.

Aspiration Mode

In this mode, the chemical reaction is carried out in a

test tube/cuvette. The instrument aspirates the reaction

mixture from the test tube/cuvette, which enters into the

flow cell (internally built reading chamber), where the

absorbance is measured. After the absorbance is measured,

the reaction mixture is passed through a different outlet

and is collected in a waste collecting bottle. As all the

measurements are done in a single flow cell, the flow cell

has to be washed after each test. Improper washing may

affect the test results of the subsequent tests. Especially, if

latex enhanced tests are used, the latex has a tendency to

stick and form a permanent coating on the internal walls of

the flow cell resulting in variation in the wavelength of the

FIG. 23.14: Effect of increasing measuring range on security range.

M-M1 = measuring range for curve 1, C1 = critical concentration

for curve 1 M-M2 = Measuring range of curve 2, C2 = critical

concentration for curve 2

FIG. 23.16: Illustrating standard curve

FIG. 23.15: Effect of increasing antibody concentration on security

range. Where 50, 75 and 100 µL are volumes of antibody and 6,000,

11,000 and 16,500 are their respective critical concentrations

714 Concise Book of Medical Laboratory Technology: Methods and Interpretations incident light, and hence, lead to erroneous results. Even

non-enhanced antibody reagents are proteinacious and

cleaning of the flow cell would remain a critical issue.

When Ag-Ab complexes are aspirated, because of the

force of aspiration, the formed immune complexes would

be structurally disturbed and may break down into smaller

complexes, which would result in lower absorbance

values. Moreover, the immune precipitates formed may

block the aspiration tube or the flow cell itself.

For turbidimetric assays designed with real sample

blanking reading principle, it would be inconvenient to run

the test in the aspiration mode. Initially, the sample mixed

with the activation buffer would have to be aspirated first

and absorbance A1 measured. Then again the activation

buffer and the sample will have to be taken in another test

tube and the principle reagent added and the reaction

read after a fixed time interval. Hence, for running one

assay twice, double the amount of activation buffer and

sample will have to be used.

Cuvette Mode

In this mode, the reaction of the reagent and sample takes

place in a measuring cuvette, and absorbance is read in

the same measuring cuvette itself. Hence, assays using

any of the reading principles can be conveniently read in

cuvette mode without wastage of reagents.

Instruments applying this mode of measuring have an

advantage. As the reactions are run in external cuvettes,

the instruments are safe from the effects of reagents.

Moreover, availability of standardized optically clean

disposable cuvettes eliminates the carryover effects of the

previous tests.