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Use of formalin-fixed, propidium iodide-stained human leukocytes as a standard for enumerating CD4+ T lymphocytes in
G M Harrison1, A J Bennett, M Moody
1Department of Medical Biochemistry and Immunology, University Hospital of Wales, Cardiff CF14 4XW, United Kingdom.
Insights
A novel, cost-effective CellBeads method precisely enumerates lymphocytes using flow cytometry. This technique offers superior accuracy and reproducibility for routine cell counting in clinical settings.
Area of Science:
- Immunology
- Biotechnology
- Analytical Chemistry
Background:
- Accurate enumeration of lymphocytes is crucial for diagnosing and monitoring various medical conditions.
- Existing methods for cell enumeration can be expensive or lack precision.
- Flow cytometry is a standard technique for cell analysis, requiring reliable internal standards.
Purpose of the Study:
- To develop and validate an inexpensive, precise method for lymphocyte enumeration.
- To introduce CellBeads as a stable and effective internal standard for flow cytometry.
- To compare the performance of CellBeads with traditional synthetic beads.
Main Methods:
- Lymphocytes were stained and fixed with propidium iodide-paraformaldehyde to create CellBeads.
- CellBeads were used as an internal standard in flow cytometry for cell enumeration.
- Whole-blood samples with known CellBeads numbers were analyzed for lymphocyte counts.
- CellBead stability and behavior during sample processing were evaluated.
Main Results:
- The CellBeads preparation was stable at 4°C for over 60 days.
- CellBeads exhibited similar behavior to normal cells during red blood cell lysis and washing.
- Lymphocyte enumerations using CellBeads as an internal standard were highly reproducible and accurate.
- The CellBeads method demonstrated superior performance compared to synthetic beads.
Conclusions:
- CellBeads provide an inexpensive, precise, and stable internal standard for lymphocyte enumeration.
- This method is suitable for routine use in clinical and research laboratories.
- The CellBeads technique enhances the accuracy and reliability of flow cytometry-based cell counting.
Abstract:
A new, inexpensive method is described that enables lymphocytes to be enumerated very precisely. Normal leukocytes were simultaneously stained and fixed with a propidium iodide-paraformaldehyde solution. The preparation obtained (CellBeads) was used as an internal standard for cell enumeration by flow cytometry and was stable at 4 degrees C for at least 60 days. Unlike synthetic beads, the CellBeads behaved similarly to normal cells during red blood cell lysis and cell washing procedures. When known numbers of CellBeads were added to whole-blood samples and the numbers of CellBeads and lymphocytes were determined, highly reproducible and accurate enumerations were obtained-far more so than when synthetic beads were used. This inexpensive method is suitable for routine use.
