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Updated: May 16, 2026
Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins
Published on: November 17, 2016
Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate
Leo Li-Ying Chan1, Daniel J Laverty, Tim Smith
1Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA 01843, USA. lchan@nexcelom.com
Insights
This study validates a new automated method for counting peripheral blood mononuclear cells (PBMCs), even with red blood cell (RBC) contamination. The acridine orange/propidium iodide (AO/PI) staining method accurately measures PBMC concentration, improving research reliability.
Area of Science:
- Immunology
- Biomedical Research
- Cell Biology
Background:
- Peripheral blood mononuclear cells (PBMCs) are crucial in immunology, oncology, and vaccine development.
- Accurate PBMC concentration and viability assessment are vital for clinical trials and research.
- Red blood cell (RBC) contamination is a common challenge in PBMC isolation, potentially causing experimental errors.
Purpose of the Study:
- To evaluate the accuracy of an automated image cytometry system for measuring PBMC concentration.
- To compare the performance of an acridine orange/propidium iodide (AO/PI) dual staining method against traditional PBMC counting techniques.
- To demonstrate the system's ability to accurately quantify PBMCs despite RBC contamination.
Main Methods:
- Compared five PBMC counting methods: manual hemacytometer, manual bright-field, manual RBC lysis, automated RBC lysis, and AO/PI dual staining.
- Utilized a fluorescence-based image cytometry system (Cellometer) for automated analysis.
- Assessed PBMC concentration and viability in samples with varying degrees of RBC contamination.
Main Results:
- All five methods yielded comparable total PBMC counts, indicating high accuracy across techniques.
- The AO/PI dual staining method with the image cytometry system accurately measured PBMC concentration.
- The automated system effectively compensated for RBC contamination in PBMC samples.
Conclusions:
- The AO/PI dual staining method is validated for accurate PBMC measurement using image cytometry.
- Automated image cytometry offers a reliable solution for PBMC quantification, even in the presence of RBC contamination.
- This technology enhances the precision and efficiency of PBMC analysis in research and clinical settings.
Abstract:
Peripheral blood mononuclear cells (PBMCs) have been widely researched in the fields of immunology, infectious disease, oncology, transplantation, hematological malignancy, and vaccine development. Specifically, in immunology research, PBMCs have been utilized to monitor concentration, viability, proliferation, and cytokine production from immune cells, which are critical for both clinical trials and biomedical research. The viability and concentration of isolated PBMCs are traditionally measured by manual counting with trypan blue (TB) using a hemacytometer. One of the common issues of PBMC isolation is red blood cell (RBC) contamination. The RBC contamination can be dependent on the donor sample and/or technical skill level of the operator. RBC contamination in a PBMC sample can introduce error to the measured concentration, which can pass down to future experimental assays performed on these cells. To resolve this issue, RBC lysing protocol can be used to eliminate potential error caused by RBC contamination. In the recent years, a rapid fluorescence-based image cytometry system has been utilized for bright-field and fluorescence imaging analysis of cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer image cytometry system has demonstrated the capability of automated concentration and viability detection in disposable counting chambers of unpurified mouse splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) under fluorescence detection. In this work, we demonstrate the ability of Cellometer image cytometry system to accurately measure PBMC concentration, despite RBC contamination, by comparison of five different total PBMC counting methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) manual counting of PBMCs in bright-field images, (3) manual counting of acetic acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The results show comparable total PBMC counting among all five methods, which validate the AO/PI staining method for PBMC measurement in the image cytometry method.
