Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis

Area of Science:

• Hematology
• Biotechnology
• Analytical Chemistry

Background:

• Traditional blood cell analysis often requires erythrocyte lysis, which can affect cell integrity and analysis of certain populations.
• Flow cytometry offers high-throughput analysis but differentiating all nucleated and non-nucleated blood cells simultaneously can be challenging.

Purpose of the Study:

• To develop and validate a multidimensional flow cytometric method for automated whole blood cell analysis without erythrocyte lysis.
• To simultaneously identify and enumerate diverse peripheral blood cell populations, including immature and nucleated cells.

Main Methods:

• Simultaneous measurement of forward light scattering, orthogonal light scattering, and fluorescence intensities of unlysed peripheral blood cells.
• Labeling cells with CD45-phycoerythrin and nucleic acid dyes LDS-751 and thiazole orange.
• Utilizing a developed software program for identification and enumeration of distinct cell populations in a five-dimensional space.

Main Results:

• Unique positions were identified for erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells.
• Platelets were accurately identified using LDS-751 staining and light-scattering properties, validated by CD41/CD42 expression.
• Quantification of major cell types correlated well with a commercial hematology analyzer, demonstrating reproducibility and stability up to 8 hours post-collection.

Conclusions:

• Multidimensional flow cytometry enables feasible automated whole blood cell analysis without erythrocyte lysis.
• Simultaneous identification of reticulocytes, nucleated erythrocytes, and immature nucleated cells is a unique capability.
• This method presents a powerful tool for assessing abnormal blood samples and advancing hematological diagnostics.