low-frequency DC measures cell volume, whereas a high-frequency electromagnetic probe

measures conductivity, an indicator of cellular internal content. the conductivity signal is

corrected for cellular volume, which yield a unique measurement called opacity. each cell also is
scanned with monochromatic laser light that reveals information about the cell surface, such as
structure, shape, and reflectivity. beckman coulter's unique rotated light scatter detection method,
which covers a 10-degree to 70-degree range, allows for separation of cell with similar volume
but different scatter characteritics. beckman coulter's newest analyzer, the unicel DxH 800, user
volume and conductivity as well as five scatter (LALS), median-angle light scatter (MALS),
lower median-angle light scatter (LMALS), and upper median-angle light scatter )UMALS).
using the data collected by the parameters listed above, the instrument applies data
transformation, the process by witch populations as well as the enhancement of sub populations
of cells. once those populations are established, a technique called the watershed concept
searches for those populations and aids in determining count as well flagging based on all the
populations found for that sample.

This combination of technologies provides a there dimensional plot of crtograph of the

WBC populations, which are separated by computer cluster analysis. Two-dimensional
scatterplots of the measurements represent different views of the cytograph. The scatterplot of
volume (y-axix) versus light scatter (x-axis) shows dear sepration of lymphocytes, monocytes,
neuorophils, and eosinophils. Basophlis are hidden be hind the lymphocytes but are seperated by
conductivity owing to their crytplasmic granulation. Single parameter histograms of volume,
conductivity, and light scatter also are available.

Two types of WBC flags (alarams or indicators of abnormality) are generated on all
hematology analyzers that provide a WBC differntial count: (1) user defined, primarily set for
distributional abnormalities, such as eosinophilia or lymphocytopenia (based on absolute
eosinophili or lympocye count ) and (2) instrument specific, primarily suspect flags for
morphologic abnormalities. For distributional flags, the user establishes reference intervals and
programs the instrument to flag each parameter as hight or low. Suspect flags indicating the
posible presence of abnormal cells are triggered when cell populations fall outside expected
regions or when spesific statistical limitations are exceeded instrument-specific suspect flags on
the coulter unicel DxH 800 system and LH 700 series include immature granulocytes/bands,
blats, variant lymphocytes, nucleated RBCs and platelet clumps. The unicel DxH800 also utilizes
the international society for laboratory hematology (ISLH) consensus rules in addition to the user
defined and system defined flags for complete data analysis in addition to theflags listed above,
inadequate separation of cell populations may disallow reporting of differential result by the
instruments and may elicit a review slide message.

The unical DxH 800 system utilizes VCS as well digital signal processing from five light
scatter angles for clear cellular resolution. On the LH 700 series , coulter utilizes an
intellikinetics application. This application is used to ensure consistency with the kinetic
reactions. It provides the instrument the best signals for analysis independent of laboratory
environment variationts. Compared with earlier models coulter intellikinetics provides better
separation of cell populations for WBCs and reticulocytes which enables better analysis by the
system algorithms.

The unicel DxH 800 also includes the number of nuleated RBCs as part of the standart
CBC report. They are identified, counted and subtracted from the white blood cell count using
volume, conductivity and the same five light scatter measurement described above. The AL2
measurement (which ferlects the amount of light absorbed as it passes through the flow cell)
initially separates the nucleated RBCs from WBCs. Algorithms are applied using the scatter
from the other angles to electronically separate and count the nucleated RBCs two scatterplots
display the nucleated RBC data by plotting axial light loss (AL2) on the x-axis againts low angel
light scatter (RLALS) and upper median angle light scatter (RUMALS) on y-axis.

Figure 15-6 represent a standart patient printout from the beckman coulter unicel DxH
800.

Sysmex Instrumentation

Sysmex corporation, formerly TOA medical electronics company,Ltd, manufactures a full line of
hematology analyzer with that provide complete RBC, platelet, and WBC analysis wth there part
differential the large XT-1800i (SF-300 and SE 9000) that performs a CBC with five part
differential and the XE series and the newest XN series that also privide a fully automated
reticulocyte count. The newest XN series modular. The series is scalable, and multiple modules
can be combined onto one platfrom. Each module contains the XN-CBC and XN-DIFF with
other option available, including XN-BF, the body fluid application. Included standard on the
CBC and DIFF modules are NRBCs, RET-He (reticulocyte hemogloblin), and IRF(immature
reticulocyte fraction). The platelet analysis on the XN also utilizes a fluorescent count, in
addition to the impedance count and optical count, called the PLT-F, performed by optical
measurement, the PLT-F can be performed on each sample or set up as a reflex based on the
laboratorys PLT criteria. The method user a fluorocell fluorescent dye (oxazine) combined with
an extended PLT counting volume and time. The PLTs can be differentiated from other cells
based on differences in tensity of the fluorescence combined with forward scattered light. The
WBC, RBC, platelete counts, hemogloblin and hematocrit are considered to be measured
directly. Three hydraulic subsystem are used for determining the hemogram: the WBC channe, in
the RBC/platelet and a separate hemogloblin channel. In the WBC and RBC transducer
chambers, diluted WBC and RBC samples are aspirated through the different apertures and
counted using the impedance (DC detection) method for counting and volumetrically sizing
cells. Two unique features enhance the impedance technology; in the RBC/platelet channel, a
sheathed stream with hydrodynamic focusing is used to direct cells through the aperture, which
reduces coincident
Figure 15-6 coulter unicel DxH 800 printout displays the CBC,DIFF and reticulocyte data for
the same patien in figure 15-7, 15-9 and 15-12. A, CBC data B, Differential with the
nucleated red blood cells (NRBCs) C, Reticulocyte data, including the IRF ( immature
reticulocte fraction) D, impedence histogram for the WBC, RBC and PLT E, Advancrd two
dimensional optical sctterplot for WBCs, NRBCs, and reticuloctes. F, suspect areain which
any sample or system flags will display.

Passage, particle volume distortion and recirculation of blood cells around

the aperture, and in the WBC and RBC/platelet channels floating thersholds
are used to discriminate each cell population.
 As cells pass througthe apertures, signals are transmitted in sequence
to the analogue circuit and particle volume distribution analysis circuits for
conversion to cumulative cell volume distribution data. Particle volume
distributiom curvesare constructed, and optimal position of the
autodiscriminationlevel (i.e,threshold) is set by the microprocessor for each
cell population. The power platelet theshold is automatical adjusted in the 2-
to 6-fl volume range, and the upper thershold is adjusted in the 12- to-30fl
range, based on particle volume distribution. Likese, the RBC lower and
upper thresholds may be set in the 25 to 75- fl and 200 to 250-fl, Volume
ranges. This floating threshold circuritry allows for discrimination of cell
populations on a sample by sample basis. Cell counts are based on pulses
between the lower and upper autpdiscriminatior levels, with dilution ratio,
volume counted and coincident passage error accounted for in the final
computergenerated numbers. In the RBC channel, the floating discriminator
is particularly useful in separating platelets froms small RBCs. The
hematocrit also is determined from the RBC/platelet channel, based on the
principle that the pulse height generated by the RBC is proportional to cell
volume. The hematocrit is the RBC cumulative pulse height and is
considered a true relative percentage volume of erythrocyte.