Related Experiment Video
Updated: Aug 8, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
A simple technique for the determination of kappa and lambda immunoglobulin light chain expression by B cells in
W M Reynolds1, A M Williamson, G J Smith
1Wessex Immunology Service, Southampton University Hospitals, UK.
Insights
A new dual fluorescence flow cytometry technique accurately analyzes surface immunoglobulin cells in whole blood. This rapid method correlates well with traditional techniques and is useful for B-cell malignancies and lymphopenia.
Area of Science:
- Immunology
- Hematology
- Analytical Chemistry
Background:
- Accurate enumeration of lymphocyte subsets is crucial for diagnosing and monitoring various hematological conditions.
- Traditional methods for cell isolation, such as density gradient centrifugation, can be time-consuming and may lead to preferential depletion of certain cell populations.
- Analyzing cell surface markers directly in whole blood samples presents challenges due to potential interference from serum factors.
Purpose of the Study:
- To develop and validate a simple, rapid, and accurate dual fluorescence flow cytometry technique for analyzing surface immunoglobulin-expressing cells in whole blood.
- To compare the efficacy of this new whole blood method with traditional density gradient centrifugation.
- To assess the applicability of the technique in various clinical scenarios, including B-cell malignancies and lymphopenia.
Main Methods:
- Development of a dual fluorescence flow cytometry protocol for direct analysis of whole blood samples.
- Comparison of cell counts obtained from the whole blood method with those from samples processed by density gradient centrifugation.
- Application of the technique to blood samples from healthy individuals and patients with B-cell chronic lymphocytic leukemia (B-CLL) and B-cell non-Hodgkin lymphoma (B-NHL).
Main Results:
- The developed whole blood flow cytometry technique demonstrated excellent correlation with results obtained from density gradient centrifugation.
- The method proved effective for analyzing B-cell malignancies, including B-CLL and B-NHL.
- Previous findings regarding the potential depletion of lymphocyte subsets by density gradient centrifugation were confirmed.
Conclusions:
- The novel whole blood dual fluorescence flow cytometry technique is a rapid, accurate, and reliable method for enumerating surface immunoglobulin-expressing cells.
- This technique offers significant advantages, including applicability in lymphopenic cases and requiring only small blood volumes.
- The method is suitable for various clinical applications, particularly when serum factors might interfere with antibody staining in whole blood analysis.
Abstract:
This article describes the development of a simple technique by which the numbers of surface immunoglobulin expressing cells can be analysed by dual fluorescence flow cytometry in samples of whole blood. We have compared the results obtained using this procedure with those obtained using samples prepared by traditional density gradient centrifugation, and demonstrate an excellent correlation between the two techniques. The method is applicable both to blood samples from normal individuals and from patients with B cell malignancies such as B-CLL and B-NHL. We have also confirmed previous findings that density gradient centrifugation may preferentially deplete certain lymphocyte subsets. This technique offers the following advantages: (i) it is rapid, (ii) it is accurate, (iii) it is reliable, (iv) it is useful in cases of lymphopenia, and (v) it requires only a small volume of blood. It is likely to be applicable to other situations in which the presence of serum factors interfere with antibody staining in whole blood.
