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10 20 Femtoliters

Figure 20.17 Coulter scatterplots of (A) leukocytes, (B) red cells, and (C) platelets.

10 20 Femtoliters

Figure 20.17 Coulter scatterplots of (A) leukocytes, (B) red cells, and (C) platelets.

immediately notice a particular deviation in numbers of and distribution of a particular cell line by analyzing scatterplots (Fig. 20.17).

How Data Are Reported

In most automated systems, the complete blood count is numerically reported. The differential is numerically recorded and then graphically displayed. These displays include scatterplots, scattergrams, and histograms. The basic principles behind the graphic displays of these data are fairly universal. Scatterplots and scattergrams place a specific cell on a grid identification system while histograms measure size thresholds of white cells, red cells, and platelets compared to the normal data for each of these cell groups.

Scatterplots and scattergrams provide colorful imaging of normal and abnormal cells. An operator can

Beckman-Coulter Instrumentation

Coulter STKS, Coulter GEN S, and Coulter LH 750 series (http://www.beckmancoulter.com) use VCS technology, which is an acronym for volume (V), conductivity (C), and laser light scatter (S). The simultaneous measurement of cell volume, conductivity, and light scatter provides high statistical accuracy. The cell volume is measured by electrical impedance using low-frequency direct current. To ensure accuracy, Coulter has incorporated pulse editing and sweep flow technology. This technology allows the cells that are being counted to line up in a single file to ensure size measurement integrity and to prevent cells from being counted twice. The RBC, WBC, and platelet counts are obtained by analyzing the number of pulses generated. The RBC, WBC, and platelet data are then plotted in the form of a histogram. The cell number is plotted on the y-axis, and the cell size is plotted on the x-axis. The MCV and RDW are derived from the RBC histogram. The MPV is derived from the platelet histogram. The HCT, MCH, and MCHC are calculated. Hemoglobin is measured by the cyanmethemoglobin method.

Conductivity is measured by using high-frequency electromagnetic current for nuclear and granular constituents. Conductivity is influenced by the internal structures of the cell such as the nuclear-to-cytoplasm ratio and the cytoplasmic granular content. A monochromatic helium:neon laser is the light source to measure light scatter for surface structure, shape, and granularity. Forward angle light scatter is affected by cell shape, surface characteristics, and cytoplasmic granular content. The enumeration of relative percentage and the absolute number of each five cells are displayed in a scatterplot.1

What Knowledge Is Necessary for the Operator of an Automated Instrument?

Operating automated cell counting instrumentation requires many skills. The operator must:

1. Know normal reference ranges.

2. Be familiar with normal scatterplots and histograms for the particular piece of equipment.

3. Be familiar with the flagging criteria determined by the particular laboratory information system (LIS).

• Reference ranges will be preset according to the LIS; specimens that fall out of the reference range are flagged.

4. Be familiar with delta checks.

• Delta checks are historical checks of test results from the patient's previous samples.

5. Be familiar with reflex testing.

• Reflex testing represents additional testing such as manual slide reviews, etc., which must be accomplished before test results can be released. Operators make decisions on which reflex tests to perform.

6. Notify the appropriate personnel of critical results.

• Critical results are those results that exceed or are markedly decreased from the reference range or the patient history of results.

7. Be familiar with daily maintenance procedures.

8. Be familiar with specimen handling and requirements.

A Sample Case Using Coulter VCS Technology

An approach to verifying and sending these results (this approach can be used for each automated system explained in this section).

1. The CBC results look normal (Fig. 20.18).

2. When we preview the results, we can see that the eosinophil count is extremely high on the differential report.

3. We also notice that the eosinophil area on the scatterplot is particularly bright.

4. The eosinophil result on the differential is flagged.

5. Delta check revealed that the patient sample had been run on the instrument 4 days before with normal results in all categories.

6. Since the abnormal results have been flagged, reflex testing demands that the best course of action is to do a manual smear review and verify the large number of eosinophils.

7. Once this is accomplished, then the results can be verified.

A Preview of Other Automated Cell Counting Instrumentation

This section will present the Sysmex and Cell-Dyne instrumentation. The Sysmex instrument uses hydro-dynamic focusing, while the Cell-Dyne instrument uses optical scatter and impedance.

Sysmex Instrumentation

Sysmex (Roche Diagnostics Corporation) manufactures a full line of hematology analyzers that include the K-4500, which provides a WBC, RBC, platelet, and three-part differential. The SE series and SF-3000 perform a CBC with a five-part differential. The newest analyzer added to the line is the XE-2100, which provides a CBC, five-part differential, and a fully automated reticulocyte count.

The SE series measures WBC, RBC, and platelets using direct current electrical impedance for counting and sizing of cells, hydrodynamic focusing, and automatic discrimination for accuracy and precision. The SE series generates the standard hematology parameters and the added parameters of RDW-SD (red cell distribution width by standard deviation), RDW-CV (red cell distribution width by coefficient), and MPV (mean platelet volume). Hemoglobin values are determined by

324 PartV • Laboratory Procedures

324 PartV • Laboratory Procedures

Figure 20.18 Coulter VCS technology.

Figure 20.18 Coulter VCS technology.

the use of a cyanide-free, nontoxic reagent and are measured at 555 nm. The white cell count uses a separate channel and utilizes DC electrical impedance.

The principle for the white cell five-part differential includes simultaneous measurements of RF and DC detection methods for separating the white cell popula tions. There are four separate detection channels for the determination of each white cell type. A plot of low-frequency DC impedance plotted on the x-axis, and high-frequency current RF on the y-axis determines lymphocytes, monocytes, and granulocytes. This channel is called the DIFF channel (Fig. 20.19). The second

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Figure 20.19 Sysmex scatterplot of WBC, lymphs, monocytes, and basophils.

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Figure 20.19 Sysmex scatterplot of WBC, lymphs, monocytes, and basophils.

channel is used with a special reagent for detecting the presence of immature cells. This channel is called the IMI channel. This channel also allows for abnormal morphology findings. The last two channels are the EO and the BASO chambers. Eosinophils and basophils are enumerated by impedance after the sample has been treated with specific lysing or buffer reagents1 (Fig. 20.20).

Cell Dyne Technology

The Cell-Dyne system (Abbott Diagnostics Instrumentation) uses three independent measurement technologies. These measurements include an optical channel for the white count and differential, an impedance channel for the red count and platelets, and a hemoglobin channel for hemoglobin determination.

A unique design of Cell DYNE is the technology of multiangle polarized scatter separation (MAPSS). The WBC count and differential are derived from this patented optical channel. A hydrodynamically focused sample stream is directed through a high-resolution flow cytometer. A cell suspension is prepared with a diluent, which maintains the WBCs in their native state, which is then passed through an air-cooled Argon ion laser light source. Scattered light is measured at multiple angles. Low-angle (1 to 3) forward light scatter represents the cell size. Wide-angle (3 to 11) forward light

Figure 20.20

Figure 20.20

326 PartV • Laboratory Procedures measures the cell complexity. Orthogonal light scatter (90) determines cell lobularity; 90 depolarized (90D) is for the evaluation of cellular granularity. Various combinations of these four angle measurements are used to differentiate the white cell populations. Neutrophils and eosinophils are separated from mononuclear cells by plotting 90 light scatter data, which are on the y-axis, and wide-angle forward light scatter data, on the x-axis. Eosinophils are separated from the neutrophil population by the eosinophils' ability to depolarize the polarized light scatter. The lymphocyte, monocyte, and basophil populations can be separated by plotting low-angle forward light scatter on the y-axis and wide-angle forward light scatter on the x-axis1 (Fig. 20.21).

Reticulocyte Counting on Automated Instrumentation

Automated reticulocyte counting is quickly becoming the standard for reticulocyte counting in clinical laboratories. For reticulocyte analysis, New Methylene Blue is incubated with whole blood samples. The dye precipi tates the basophilic RNA network found in reticulocytes. Hemoglobin and unbound stain are removed by adding a clearing reagent, leaving clear spherical mature RBCs and darkly stained reticulocytes. Stained reticulo-cytes are differentiated from mature cells and other cell populations by light scatter, direct current measurements, and opacity characteristics. The normal reference range is 0.5% to 1.5%. In comparison to the manual reticulocyte count in which 1000 red cells are counting, the automated reticulocyte procedure counts 32,000 red cells.

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