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Resolving leukocytes using axial light loss.

C C Stewart¹, S J Stewart, R C Habbersett

¹Los Alamos National Laboratory, Cell Biology Group, New Mexico 87545.

|July 1, 1989

Summary

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Axial light loss (ALL) combined with CD45 antibody offers a robust method for blood cell differential analysis. This technique effectively distinguishes cell types and identifies dead cells, even in fixed samples, enhancing safety and efficiency.

Area of Science:

Hematology
Immunology
Analytical Chemistry

Background:

Axial light loss (ALL) measures light lost from a laser beam by particles.
CD45 is a key marker for distinguishing leukocyte populations.

Purpose of the Study:

To evaluate the utility of Axial Light Loss (ALL) in combination with CD45 for blood cell analysis.
To demonstrate the effectiveness of ALL in resolving various leukocyte subsets and dead cells.

Main Methods:

Utilizing a single laser flow cytometer with three-color fluorescence.
Employing Axial Light Loss (ALL) measurements alongside CD45 antibody.
Incorporating fluoresceinated CD45, phycoerythrin-labeled NKH1, and biotinylated CD3 with DuoCHROME.

Main Results:

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ALL and CD45 effectively resolve lymphocytes, monocytes, granulocytes, and dead cells.
Degranulated granulocytes are resolved using ALL vs. CD45, not traditional scatter methods.
A complete blood differential is achievable, including T-cell and B-cell identification, and NK cell enumeration.

Conclusions:

Axial light loss (ALL) provides a reliable method for blood cell differentiation and dead cell detection.
The use of ALL eliminates the need for propidium iodide for dead cell identification.
Sample fixation is feasible with ALL analysis, reducing biohazard risks.