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[Narrator:] Rubella testing in the small hospital laboratory.

Serology laboratories, both large and small,

are constantly being deluged with requests for rubella antibody testing.

Technology is now available for determination

of immune status to rubella, diagnosis of postnatal rubella, and diagnosis of congenital rubella.

We will discuss several of the standardized and clinical methods available,

along with the clinical settings in which each would be appropriate.

In school-age children, rubella, or German measles,

is usually a self-limiting disease characterized by

upper respiratory involvement, lymphoadenopathy, and an erythematous rash.

To determine if an acute infection is in progress,

the most widely used procedure is the hemagglutination inhibition, or HAI.

Hemagglutination inhibition is based on

the serological principle that the agglutination

of red blood cells by rubella antigen can be blocked

or inhibited by antibody and a serum specificfor that antigen.

A treatment process is used to remove serum inhibitors, which are not

specific rubella antibody.

By testing ever-decreasing dilutions

of a serum against a constant amount of antigen,

the antibody content of the serum can be determined.

Complications of the technique are one,

non-specific inhibitors of agglutination

may exist in normal serum, which block

the viral hemagglutination by obscuring sites

either on the red blood cell or on the virus.

Test serum may contain hemagglutinating substances.

To be truly diagnostic for acute rubella infection,

an established format should be followed for drawing the HAI test specimens.

One, the HAI test must be run on paired sera.

Acute serum should be taken early in the infection,

preferably no later than three days after clinical onset.

A second specimen should be drawn between two to three weeks after the onset of clinical symptoms.

Two, the paired sera are then assayed in the same test run.

Three, a four-fold or greater rise in titer is diagnostic of recent rubella infection.

There is considerable variability in the antibody titers maintained during life.

Therefore, a diagnosis cannot be made on a single serum sample.

Four, stable or falling titer can only be interpreted as infection in recent past.

When congenital infection is suggested in a neonate, paired sera should be taken as follows.

One. One specimen is drawn from both the mother and the infant

when the infant is less than six months of age.

Two, a second specimen is taken from both the infant and mother

when the infant is 6 to 12 months old.

If when performing hemagglutination inhibition testing, 

the rubella antibody of the infant is present in a stable titer amount,

possible congenital infection is indicated.

Also, if the mother does not have rubella antibodies or has a very low titer, 

it is unlikely that the baby had congenital infection.

A second approach to determining possible congenital infection

is to demonstrate rubella-specific IgM antibody in the infant serum.

Such IgM is present only in primary rubella infection.

It is short-lived and begins to decrease a few weeks after the infection.

The immunoglobulin in serum is separated by a sucrose density gradient, 

and the rubella-specific IgM is then measured by HAI.

Only when rubella-specific IgM is found can one be sure of recent rubella infection.

Passive hemagglutination, or PHA, is another rubella testing procedure

which has been standardized and is routinely used in the serology laboratory.

It is easily performed and much quicker than hemagglutination inhibition.

Passive hemagglutination will begin picking up antibody levels about two weeks later than the HAI.

A positive PHA indicates that the serum sample has HAI titer of 1 to 8 or greater.

Passive hemagglutination is used primarily as a screening technique.

It allows for an easy and quick method for determination of immune status.

PHA tests that are positive indicate that the patient has a protective level of rubella antibody.

If the PHA test is negative, it should be repeated by HAI.

If the HAI titer is less than 1 to 8, the patient has insufficient rubella antibody to protect him from infection.

Women of childbearing age who are sero-negative should be vaccinated with live virus vaccine.

Due regard should be taken for the potential dangers of vaccination during pregnancy.

Many physicians determine the rubella antibody status of pregnant women at the patient's first prenatal visit.

Those without antibody are monitored through early pregnancy for sero conversion.

The passive hemagglutination test incorporates rubella antigen onto erythrocytes,

which will agglutinate in the presence of rubella antigen in the patient's serum.

The test is performed in the V-bottom microtiter plate.

If the patient has rubella antibody, the agglutinated antigen antibody complex

settles to the bottom of the V-shaped plate in a dispersed pattern.

If little or no agglutination has occurred, the red cells slide to the bottom,

forming a sharp compact button.

The V-plate is read by using a mirrored microtiter plate reader.

In conclusion, there are many sophisticated techniques

for rubella testing available on the market, some old, some new.

Some techniques used other than the ones mentioned here are

radioimmunoassasy, enzyme-linked immunosorbent assay, fluorometry orIFA, and hemolysin-in-gel.

These tests may grow to be the techniques of the future,

but because of their standardization, hemagglutination inhibition, or HAI,

and passive hemagglutination, PHA,

should be the first choice in rubella immunity screening

until the superiority of other methods has been proven.

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[Mark-Maris]

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