Serology/Immunology 607

4. A decrease in thyrotropin values has been reported

with the administration of propranolol, methimazol,

dopamine and d-thyroxine.

5. Genetic variations or degradation of intact TSH into

subunits may affect the biding characteristics of the

antibodies and influence the final result. Such samples

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

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

In addition, the RLU’s of the calibrators have been normalized to

approximately 100,000 RLU’s for the A calibrator (greatest light output).

This conversion eliminates differences caused by efficiency of the

various instruments that can be used to measure light output.

FIG. 22.24: Example showing average RLU intersects calibration curve

at TSH concentration

Note 1: Computer data reduction software designed for

chemiluminescence assays may also be used for the data

reduction. Duplicates of the unknown may be averaged as

indicated (Fig. 22.24).

Note 2: Monobind can assist the laboratory in the purchase

and implementation of equipment/software to measure

and interpret chemiluminescence data.

QC Parameters

1. The Dose Response Curve should be within established parameters.

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

established ranges.

Limitations of Procedure

A. Assay performance

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

is held constant for reproducible results. Pipetting of

samples should not extend beyond ten (10) minutes

to avoid assay drift. If more than one (1) plate is used,

it is recommended to repeat the dose response curve.

Failure to remove adhering solution adequately in the

aspiration or decantation wash step(s) may result in

poor replication and spurious results.

2. Sample(s), which are contaminated microbiologically,

should not be used in the assay. Highly lipemeic or

hemolyzed specimen(s) should similarly not be used.

3. Patient specimens with TSH concentrations above 40

µIU/mL may be diluted with the zero calibrator and

reassayed. The sample’s concentration is obtained by

multiplying the result by the dilution factor.

4. Each component in one assay should be of the same

lot number and stored under identical conditions.

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.

608 Concise Book of Medical Laboratory Technology: Methods and Interpretations normally exhibit different results among various

assay systems due to the reactivity of the antibodies

involved.

“Not intended for newborn screening”

Expected Ranges of Values

A study of euthyroid adult population was undertaken to

determine expected values for the TSH CIA Microplate

Test System. The number and determined range are given

in Table 22.2. A nonparametric method (95% Percentile

Estimate) was used.

It is important to keep in mind that expected values

for normal population is dependent upon a multiplicity

of factors: The specificity of the method, the population

tested and the precision of the method in the hands of

the analyst. For these reasons each laboratory should

depend upon the range of expected values established

by the Manufacturer only until an in-house range can

be determined by the analysts using the method with a

population indigenous to the area in which the laboratory

is located.

TABLE 22.2: Expected values for the TSH CIA test system (in µIU/mL)

Number 139

Low normal range 0.39

High normal range 6.16

70% confidence intervals for 2.5 percentile

Low range 0.28–0.53

High range 5.60 – 6.82

TESTS FOR SYPHILIS

VDRL Test

Conventional VDRL Test

Serological testing, for the diagnosis of syphilis, traditionally has been based on the detection of “Reagin” by the use

of antigen, prepared from normal tissues, most commonly

beef heart. VDRL slide test can be used both qualitatively

and quantitatively for the detection of “Reagin” in serum.

As other better methods are available, this method is hardly even used.

Principle

A phospholipid viz. cardiolipin, derived from beef heart

muscle together with cholesterol and lecithin, is used as

an antigen. After mixing the antigen with patient’s serum,

the reaction is accelerated by rotatory agitation either

on a mechanical shaker or by hand. The antigen reacts

with reagin and forms floccules. These floccules can be

observed with naked eye, hand lens or under a low power

objective of a microscope when the reaction is weak.

Equipment Required

1. VDRL Test Slide: A 2” × 3” glass slide with 12 paraffin

or ceramic rings of approximately 14 mm inside

diameter.

2. Hypodermic needles without bevels (18, 19 gauge).

3. Syringe (1–2 mL).

4. Thirty mL flat or concave inner bottomed glass

stoppered, narrow mouth bottle, approximately 35

mm in diameter (bottles with convex inner bottom

surface are unsatisfactory).

Precautions

1. Use clean and dry glassware.

2. Allow all reagents and samples to reach room

temperature before starting the test.

3. Carry out the test at room temperature (preferably

between 23 and 29°C).

Sample

Serum (0.05 mL is required for qualitative test and 0.1 mL

is needed for quantitative test.