POLYMERASE CHAIN REACTION & ITS CLINICAL APPLICATIONS TO BLOOD BANKING

Hawazin Faruki, Dr.P.H., Director Molecular Diagnostics Reference Laboratory
and
Alan Winkelstein, M.D., Medical Director

INTRODUCTION

The polymerase chain reaction (PCR) is an extraordinarily sensitive molecular-based diagnostic test that potentially has widespread use in clinical medicine.  One of its first applications is in the early and definitive recognition of infectious diseases such as HIV.  For example, HIV-I PCR based assays can recognize as few as one HIV-1 infected cell admixed with 10⁵ uninfected cells.

PRINCIPLES OF PCR TESTING

The PCR assay for HIV is performed by lysing human nucleated cells containing integrated HIV virus genome thereby releasing human as well as viral double stranded DNA into solution.  The DNA strands are then separated from each other by heating (see figure).

Two unique DNA primers are added.  In the case of HIV, these primers are complementary to an area of the proviral genome coding for the gag protein.  Primers bind only when the complementary sequences are present, thus this step determines the assay’s specificity.  Following binding, the primers are elongated by sequential addition of nucleotides complementary to those present on the native DNA strand through a catalytic reaction employing an enzyme, taq polymerase.

The resulting double stranded DNA segments, consisting each of a strand of native DNA and a strand synthesized fro the primer, are again separated by heating.  Upon cooling, the primers again bind specifically to the single stranded DNA sequences of interest initiating the taq polymerase to catalyze the elongation from the primer site.

This process of heating to separate DNA strands, primer binding and DNA elongation constitutes a cycle and is repeated up to 30 or more times.  Thus, one section of a gene is repeatedly duplicated, resulting in the production of millions of copies.  These are all replicates of the original sequence and are in sufficient concentrations that can be detected by either radioisotopic labeling procedures or a colorimetric reaction.

One of the major problems with the initial PCR based tests was the high incidence of false positive reactions, which occurred because of trace amounts of cross contamination.  The assay is so powerful that aerosolized contamination of a negative specimen by even a single copy of a gene may yield a positive test.  This problem has a been largely circumvented by the addition of new control procedures.

HIV-I Testing

The utility of PCR testing for HIV-I has already been well established for the following situations:

1. Neonates born to HIV-I positive mothers.

Invariably, these infants will have a positive HIV-I antibody test because of maternal transmission of antibodies.  However, only 12 to 30 percent are actually infected.  PCR testing, which measures the presence of the virus, not the antibody, can be used to determine neonatal infection at birth.

2. Individuals with positive screening tests and indeterminate Western blot results. 

In many instances, these individuals are not infected with HIV-1.  However, with conventional antibody testing, this can be established only after prolonged periods of observation.

3. Individuals who are in the “window period”, the time between infection and seroconversion.

Some of these individuals may present with a mononucleosis-like illness due to an acute HIV-I infection.

4. A health care worker who experiences an accidental exposure of blood or blood-containing fluids from an HIV-I positive patient. 

Although the risk of infection is low, a small percentage do become infected.  This possibility is a source of great anxiety and conventional serologic studies are unable to exclude infection for at least six months.  PCR testing can detect the presence of the virus in mononuclear cells, probably within two weeks after exposure.  Thus, these results can provide exposed workers with a definitive information early, thereby alleviating prolonged periods of uncertainty and anxiety.

other potential testing

PCR-based procedures may also be utilized to detect many other infectious diseases including hepatitis C, a major cause of chronic liver disease.  It is well known that in low risk populations, such as blood donors, a high percentage of HCV reactive subjects have false positive tests.  Likewise, patients with acute hepatitis may not develop positive antibody tests for several months after infection. 

Early diagnosis of tuberculosis may be facilitated by PCR testing as well.  Primers for Myobacterium tuberculosis and other forms of mycobacteria can rapidly detect organisms in body fluids.  By contrast, results from conventional culture are generally not available for two to six weeks.  PCR may also be useful for recognizing drug resistant strains of M. tuberculosis, a medical problem of growing importance. 

Other infectious disease applications of PCR include the detection of Borrelia burgdorferi, the causative agent for Lyme disease, human papilloma virus, a suspected cause of cervical cancer, cytomegalovirus, chlamydia, and hepatitis B.  PCR can distinguish between HTLV-I and II infections, which serologically are indistinguishable.

PCR is also finding numerous applications in both oncology and in genetic counseling.  As DNA abnormalities for specific forms of cancers are identified, primers can be developed that provide sensitive diagnostic information and detect minimal residual disease after potentially curative therapy.  In genetics, numerous inherited disorders can be detected in utero. 

Copies of the Transfusion Medicine Update can be obtained by contacting Deborah Small at (412) 209-7320 or
by e-mail:  dsmall@itxm.org.