Proposed reference method for peripheral-blood monocyte counting using fluorescence-labelled monoclonal antibodies

Area of Science:

• Hematology
• Immunophenotyping
• Clinical Diagnostics

Background:

• Automated hematology analyzers require accurate reference methods for monocyte counting.
• Previous studies have explored flow cytometry for monocyte enumeration, but comparisons of different techniques are limited.

Purpose of the Study:

• To compare various flow cytometric techniques against manual differentials and an automated hematology analyzer for monocyte counting.
• To evaluate the potential of flow cytometry as a new reference method for monocyte enumeration.

Main Methods:

• Relative monocyte counts were determined using a Coulter Profile II flow cytometer with two CD45-FITC/CD 14-PE antibody combinations.
• Two whole-blood lysis techniques, including a no-wash method, were employed.
• Results were compared to a 600-cell manual differential and a Coulter STKS hematology analyzer.

Main Results:

• All flow cytometric methods showed excellent correlation (r >= 0.925) with manual differentials, with no significant bias.
• Flow cytometry precision significantly surpassed that of manual and automated methods.
• Excellent correlation (r >= 0.995) was observed between flow cytometric techniques, with identical mean monocyte counts.

Conclusions:

• Flow cytometry with fluorescently labeled monoclonal antibodies is a highly precise and accurate method for monocyte counting.
• It shows strong potential as an ideal new reference method for evaluating hematology analyzer performance.
• Standardization of sample preparation procedures is crucial for establishing flow cytometry as a robust reference method.