PN 177196BB
7-8
DATA MANAGEMENT AND REVIEW
DATA ANALYSIS - AN OVERVIEW OF THE DILUTIONS AND MEASUREMENTS
Dilution Used to Determine LYMPH, MONO, NEUT, and EO Counts and a Secondary WBC Count
1. Two dilutions are required to obtain differential parameter results:
a. WBC/BASO dilution
b. DIFF dilution
2. The final dilution in the DIFF bath is used to determine lymphocytes, monocytes,
neutrophils, and eosinophils counts and a secondary WBC count.
3. The final dilution inside the DIFF bath is prepared in two stages.
a. First stage: 25 µL of whole-blood is delivered to DIFF bath in a flow of Fix reagent.
1) Dilution is incubated for 12 seconds, allowing the Fix reagent to lyse RBCs,
stabilize WBCs in their native forms, and differentially stain lymphs, monos,
neutrophils, and eos, with eosinophils staining most intensely.
2) The Analyzer maintains the reagents and reaction at a regulated 35°C (95°F).
b. Second stage: 1.0 mL of Diluent is added to the DIFF bath to stop the cytochemical
reaction and form the final 1:80 dilution.
4. The final 1:80 dilution is transferred to the flow cell where each cell is measured for its
impedance (volume) and absorbance (optical detection of cytochemistry changes).
5. The final 1:80 dilution is analyzed in the flow cell to:
a. Develop the DiffPlot from which four of the five WBC populations are determined:
lymphocytes, monocytes, neutrophils, and eosinophils.
Note: In a typical whole-blood sample, the basophil population (determined in the
WBC/BASO bath) is very small compared to the other four WBC populations.
b. Determine a secondary WBC count that is compared with the reference WBC count
determined in the WBC/BASO bath; if WBC count results from the two
methodologies exceed predefined limits, the WBC count result is flagged.
6. Table 7.2-4 summarizes the dilutions made and the measurement methods used to
obtain the DiffPlot data. This same information is covered in
Table 2.3-5, Technical
Characteristics for Acquisition of the DiffPlot,
in the Online Help System or the Instructions for
Use manual.
Table 7.2-4 Technical Characteristics for Obtaining the DiffPlot
Dilution Characteristics Volume of whole-blood 25 µL
Volume of Fix 1000 µL
Volume of Diluent 1000 µL
Final dilution ratio 1:80
Reaction temperature 35°C (95°F)
Incubation duration 12 seconds
Measurement Characteristics Methods of analysis Coulter Principle and Optical
Transmission with
hydrofocusing
Aperture diameter 60 µm
Diameter of the flow 42 µm
Volume injected 72 µL
Injection duration 15 seconds
Data accumulation 12 seconds
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