Biomedical Engineering Reference
In-Depth Information
Fig. 3.4
(
a
) Histograms of detected CD4 cells in whole blood (dilution 5:1) as a function of
fluorescent intensity. This illustrates an absolute CD4 count in whole blood (no lyse, no wash,
dye: PE) (Adapted from [
30
]). (
b
) CD4% measurement obtained from whole blood (no lysing)
with PARC's spatial modulation technique. The pattern agrees in all essential features with that
from a commercial BD FACSCount instrument (Adapted from [
30
])
a human myelomonocytic antigen that is present on the majority of granulocytes.
No lysing of the red blood cells or washing steps to separate the tagged blood
cells from unbound dye were used. We have tested a variety of blood samples with
different dilutions (1:10 to 1:1 blood to buffer ratio), incubation times (10-40 min),
and temperatures (RT, 37
ı
C).
A histogram of detected CD4 cells in whole blood (no lyse, no wash, dye: PE) is
shown in Fig.
3.4
a as a function of fluorescent intensity. Sample preparation was as
follows: 25 l whole blood, 2l CD4-PE, and 123l PBS, dilution of 1:5. This
illustrates a CD4 count in whole blood (no lyse, no wash, dye: PE). The plots
exhibit two peaks that are attributed to CD4 lymphocytes (right peak) and CD4
monocytes (left peak). This histogram is representative for many measurements on
this donor blood (i.e., for repeated measurements on the same sample and samples
with modified sample preparation). The average absolute CD4 count was
1;800
CD4 cells per l blood with a variation of about
6 %. The recorded CD4 count
is at the upper end but within the expected range for human blood. The relative
count rate of lymphocytes and monocytes and, more importantly, the peak distance
(intensity ratio) are in good agreement with data reported in the literature [
31
].
Measurements of %CD4 were conducted on samples of whole blood that
were stained with BD CD4% reagent (BD#339010). For this measurement, two
fluorescence signals were simultaneously recorded from the same detection area.
They were recorded from opposite sides of the fluidic chip. A volume of 30l
of analyte containing 3 l of whole blood (specified protocol for the CD4%
reagent) was analyzed within 5 min. The sample was prepared by following the
recommended BD sample preparation protocol to obtain samples with a dilution of
1:10.
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