Biomedical Engineering Reference
In-Depth Information
enumerations. Hybridoma technology led the way to refining the production of
antibodies with higher specificity. The use of highly specified monoclononal
antibodies complements the rapid throughput of flow cytometry. While manual
counting methods are still an option, flow cytometry for its reliability and ease
of operation has become the predicate technique for the quantitative analysis of CD4
T- l ymphocy t es .
Absolute values of CD4 positive cells can be calculated using a two-system
approach, combining the lymphocyte counts obtained from a hematology analyzer
and the percentage value from a flow cytometer. However, statistical error inherent in
this dual platform method can contribute to variability in the determination of CD4
absolute values. Alternatively, the use of standardization beads or a fixed volume of
specimen in a single platform approach allows a more direct measurement of the
absolute marker cell count in blood samples. The single platform approach has gained
wide acceptance as the preferred method in performing CD4 enumeration when
monitoring HIV/AIDS patients. The manual method for CD4 enumeration, however
overshadowed by automation and instrumentation development, has maintained its
utility particularly in resource-limited settings. The Manual CD4 Kit from Beckman
Coulter consists of CD14 labeled cytospheres, CD4 labeled cytospheres, and an
erythrocyte-lysing reagent.Whole blood collected in EDTA is initially incubatedwith
CD14 reagent to block monocyte binding and then labeled with CD4 conjugated
cytospheres. Lysing solution is added to this mixture and this preparation is loaded
onto a hemocytometer for counting by light microscopy. CD4 positive cells are
identified by the presence of three or more large latex spheres attached to the cell
surface (Figure 8.3). Monocytes react with the CD14 reagent to yield a brown
coloration and are excluded from the analysis. The CD4 รพ cells are counted and
reported as cells/
L.
To date, there have been two flow cytometry-based systems marketed that are
dedicated to single-platform CD4 absolute counting, Becton Dickinson (San Jose,
CA) and Guava Technologies (Millipore Corporation, Billerica, MA). The Becton
Dickinson FACSCount system introduced in 1994 uses counting beads to directly
measure the absolute values of CD3, CD4, and CD8 populations. Recent revisions to
m
FIGURE 8.3 Rosetting cytospheres (from Frank Mandy, National Laboratory for HIV
Immunology (NHLI), Public Health Agency of Canada, Ottawa, ON). (See the color version
of the figure in the Color Plates section.)
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