Microbiology, 2021

846 20 • Laboratory Analysis of the Immune Response
CHECK YOUR UNDERSTANDING
• What is the purpose of the laser in a flow cytometer?
• In the output from a flow cytometer, the area under the histogram is equivalent to what?
Clinical Focus
Resolution
After notifying all 1300 patients, the hospital begins scheduling HIV screening. Appointments were
scheduled a minimum of 3 weeks after the patient’s last hospital visit to minimize the risk of false
negatives. Because some false positives were anticipated, the public health physician set up a counseling
protocol for any patient whose indirect ELISA came back positive.
Of the 1300 patients, eight tested positive using the ELISA. Five of these tests were invalidated by negative
western blot tests, but one western blot came back positive, confirming that the patient had indeed
contracted HIV. The two remaining western blots came back indeterminate. These individuals had to
submit to a third test, a PCR, to confirm the presence or absence of HIV sequences. Luckily, both patients
tested negative.
As for the lone patient confirmed to have HIV, the tests cannot prove or disprove any connection to the
syringes compromised by the former hospital employee. Even so, the hospital’s insurance will fully cover
the patient’s treatment, which began immediately.
Although we now have drugs that are typically effective at controlling the progression of HIV and AIDS,
there is still no cure. If left untreated, or if the drug regimen fails, the patient will experience a gradual
decline in the number of CD4 helper T cells, resulting in severe impairment of all adaptive immune
functions. Even moderate declines of helper T cell numbers can result in immunodeficiency, leaving the
patient susceptible to opportunistic infections. To monitor the status of the patient’s helper T cells, the
hospital will use flow cytometry. This sensitive test allows physicians to precisely determine the number of
helper T cells so they can adjust treatment if the number falls below 500 cells/µL.
Jump to the previous Clinical Focus box.
Cell Sorting Using Immunofluorescence
The flow cytometer and immunofluorescence can also be modified to sort cells from a single sample into
purified subpopulations of cells for research purposes. This modification of the flow cytometer is called a
fluorescence-activated cell sorter (FACS). In a FACS, fluorescence by a cell induces the device to put a charge
on a droplet of the transporting fluid containing that cell. The charge is specific to the wavelength of the
fluorescent light, which allows for differential sorting by those different charges. The sorting is accomplished
by an electrostatic deflector that moves the charged droplet containing the cell into one collecting vessel or
another. The process results in highly purified subpopulations of cells.
One limitation of a FACS is that it only works on isolated cells. Thus, the method would work in sorting white
blood cells, since they exist as isolated cells. But for cells in a tissue, flow cytometry can only be applied if we
can excise the tissue and separate it into single cells (using proteases to cleave cell-cell adhesion molecules)
without disrupting cell integrity. This method may be used on tumors, but more often, immunohistochemistry
and immunocytochemistry are used to study cells in tissues.
LINK TO LEARNING
Watch videos to learn more about how flow cytometry (https://openstax.org/l/22flowcytometry) and a FACS
(https://openstax.org/l/22FACSwork) work.
Access for free at openstax.org.