Note: Do not use the working substrate if it looks blue.

Test Procedure

Before proceeding with the assay, bring all reagents,

serum references and controls to room temperature

(20–27°C).

1. Format the microplates’ wells for each serum

reference, control and patient specimen to be assayed

in duplicate. Replace any unused microwell strips

back into the aluminum bag, seal and store at 2–8°C.

2. Pipette 0.050 mL (50 µL) of the appropriate serum

reference, control or specimen into the assigned well.

3. Add 0.100 mL (100 µL) of the TSH Enzyme Reagent

to each well. It is very important to dispense all

reagents close to the bottom of the coated well.

4. Swirl the microplate gently for 20–30 seconds to mix

and cover.

5. Incubate 60 minutes at room temperature.**

6. Discard the contents of the microplate by decantation

or aspiration. If decanting, tap and blot the plate dry

with absorbent paper.

7. Add 300 µL of wash buffer (see Reagent Preparation

Section) decant (tap and blot) or aspirate. Repeat two

(2) additional times for a total of three (3) washes.

An automatic or manual plate washer can be used.

Follow the manufacturer’s instruction for proper

usage. If a squeeze bottle is employed, fill each well

by depressing the container (avoiding air bubbles) to

dispense the wash. Decant the wash and repeat two

(2) additional times.

8. Add 0.100 mL (100 µL) of working substrate solution

to all wells (see Reagent Preparation Section). Always

add reagents in the same order to minimize reaction

time differences between wells.

 Do not shake the plate after substrate addition.

9. Incubate at room temperature for fifteen (15)

minutes.

10. Add 0.050 mL (50 µL) of stop solution to each well

and mix gently for 15–20 seconds. Always add

reagents in the same order to minimize reaction

time differences between wells.

11. Read the absorbance in each well at 450 nm (using

a reference wavelength of 620–630 nm to minimize

well imperfections) in a microplate reader. The

results should be read within thirty (30) minutes of

adding the stop solution.

Serology/Immunology 601

** For better low-end sensitivity (< 0.5 µIU/mL). Incubate

120 minutes at room temperature. The 40 µIU/mL

calibrator should be excluded since absorbance over 3.0

units will be experienced. Follow the remaining steps.

Quality Control

Each laboratory should assay controls at levels in the low,

normal, and high range for monitoring assay performance.

These controls should be treated as unknowns and values

determined in every test procedure performed. Quality

control charts should be maintained to follow the

performance of the supplied reagents. Pertinent statistical

methods should be employed to ascertain trends. The

individual laboratory should set acceptable asssay

performance limits. Other parameters that should be

monitored include the 80, 50 and 20% intercepts of the

dose response curve for run-to-run reproducibility. In

addition, maximum absorbance should be consistent with

past experience. Significant deviation from established

performance can indicate unnoticed change in experimental

conditions or degradation of kit reagents. Fresh reagents

should be used to determine the reason for the variations.

Results

A dose response curve is used to ascertain the concentration

of thyrotropin in unknown specimens.

1. Record the absorbance obtained from the printout

of the microplate reader as outlined in following

example (An example of the 120-minute incubation

is presented in italic type).

2. Plot the absorbance for each duplicate serum reference

versus the corresponding TSH concentration in

µIU/mL on linear graph paper (do not average the

duplicates of the serum references before plotting).

3. Draw the best-fit curve through the plotted points.

4. To determine the concentration of TSH for an

unknown, locate the average absorbance of the

duplicates for each unknown on the vertical axis of

the graph, find the intersecting point on the curve,

and read the concentration (in µIU/mL) from the

horizontal axis of the graph (the duplicates of the

unknown may be averaged as indicated). In the

following example, the average absorbance (1.019)

intersects the dose response curve at (15.3 µIU/mL)

TSH concentration (Fig. 22.23).

QC Parameters

In order for the assay results to be considered valid the

following criteria should be met:

1. The absorbance (OD) of calibrator 0 ng/dL should be

> 1.3.

2. Four out of 6 quality control pools should be within

the established ranges.

*The data presented above are for illustration only and should not

be used in lieu of a dose response curve prepared with each assay.

FIG. 22.23: Example showing average absorbance intersects dose

response curve at TSH concentration

602 Concise Book of Medical Laboratory Technology: Methods and Interpretations Limitations of Procedure

A. Assay performance

1. It is important that the time of reaction in each well is

held constant for reproducible results.

2. Pipetting of samples should not extend beyond ten

(10) minutes to avoid assay drift.

3. If more than one (1) plate is used, it is recommended

to repeat the dose response curve.

4. Addition of the substrate solution initiates a kinetic

reaction, which is terminated by the addition of the stop

solution. Therefore, the addition of the substrate and the

stopping solution should be added in the same sequence

to eliminate any time-deviation during reaction.

5. Plate readers measure vertically. Do not touch the

bottom of the wells.

6. Failure to remove adhering solution adequately in the

aspiration or decantation wash step(s) may result in

poor replication and spurious results.

7. Use components from the same lot. No intermixing of

reagents from different batches.

8. Highly lipemic, hemolyzed or grossly contaminated

specimen(s) should not be used.

B. Interpretation

1. If computer controlled data reduction is used to

interpret the results of the test, it is imperative that the

predicted values for the calibrators fall within 10% of

the assigned concentrations.

2. Serum TSH concentration is dependent upon a

multiplicity of factors: Hypothalamus gland function,

thyroid gland function, and the responsiveness of

pituitary to TRH. Thus, thyrotropin concentration

alone is not sufficient to assess clinical status.

3. Serum TSH values may be elevated by pharmacological

intervention. Domperiodone, amiodazon, iodide,

phenobarbital, and phenytoin have been reported to

increase TSH levels.