Biomedical Engineering Reference
In-Depth Information
channels. The identifier channel has high SNR, so we can use the identifier channel
to produce the template for correlation with the reporter channel. That is, the
software detects particles by looking for a strong peak in the power spectrum of
the identifier channel and then correlates the detected time-domain signal from
the identifier channel, normalized for total intensity, with the reporter channel to
measure reporter intensity. There is no need to compute the full convolution because
the two channels are exactly synchronized.
3.2.2.5
Multiplexed Mask
Also as described below, we have also measured two differently tagged types of
particles with a single read-out channel, by using a multiplexed mask modulated
at one spatial frequency for one color and at another spatial frequency for another
color. The two frequencies are at a known ratio of r. For this case, the algorithm
detects particles by looking for a strong peak in the power spectrum and then
classifies the particle by checking the power levels of frequencies r and 1/r times
the detected peak. This algorithm gave error rate about 1 %, with the few errors
resulting from overlapping signals.
3.2.3
CD4 Count in Whole Blood
The spatial modulation technique has been extensively evaluated with measure-
ments of absolute CD4
and percentage CD4 counts in human blood, which are
required for screening, initiation of treatment, and monitoring of HIV-infected
patients. And the technique has been benchmarked against a commercial instrument
(BD FACSCount) with a direct one-to-one comparison of measurements on the
same labeled blood samples, with excellent agreement for both absolute CD4 and
CD4% as discussed in [
30
].
The evaluation was performed with a prototype bench-top instrument capable
of measuring absolute CD4, CD8, and percentage CD4 in whole blood. A sample
of tagged blood could be analyzed in less than 5 min. Sample preparation required
only simple dilution, mixing, and incubation steps, with no lysing or washing step.
The mixing was performed by repeatedly pipetting the sample-analyte mixture into
a vial.
Measurements of CD4 were conducted on samples of whole blood. The samples
were prepared with the standard BD CD4 reagent (PE-CD4, PE/CY5-CD3, and
known number of fluorescent microbeads) and a recently introduced FACSCount
CD4% reagent kit (BD#339010) that consists of a single tube containing
a mixture of the following: three monoclonal antibodies CD4/CD14/CD15
(conjugated with PE/PE-Cy5/PE-Cy5, respectively), a nucleic acid dye, and a
known number of fluorescent microbeads. The antibody to CD14 recognizes a
human monocyte/macrophage antigen, whereas the antibody to CD15 recognizes
C
