Optimizing PBMC Cryopreservation and Utilization for ImmunoSpot^® Analysis of Antigen-Specific Memory B Cells

Area of Science:

• Immunology
• Cellular immunology
• Diagnostic assay development

Background:

• Assessing antigen-specific memory B cells (Bmem) is crucial for predicting antibody responses.
• Current B cell ImmunoSpot® assays require large blood volumes, limiting clinical applications.
• Standard assays hinder routine immune diagnostics and clinical trial inclusion.

Purpose of the Study:

• To develop strategies for reducing blood volume requirements for B cell ImmunoSpot® assays.
• To enable reliable Bmem frequency assessment with fewer peripheral blood mononuclear cells (PBMCs).
• To enhance the feasibility of Bmem characterization in larger patient cohorts.

Main Methods:

• Reduced cell numbers to 2-3 million PBMCs per antigen from 2-3 mL of blood.
• Employed serial dilution in singlet wells for Bmem frequency assessment.
• Multiplexed B cell ImmunoSpot® assays for simultaneous detection of Ig classes or IgG subclasses.

Main Results:

• Reliable Bmem frequency assessments were achieved with significantly fewer PBMCs.
• Multiplexing assays did not compromise sensitivity for Ig class or subclass detection.
• Cryopreserved PBMCs retained functionality, equivalent to fresh samples.
• Reduced cell requirements allow for freezing fewer PBMCs per vial, addressing sample availability issues.

Conclusions:

• Optimized B cell ImmunoSpot® assays require less blood, increasing clinical feasibility.
• Cryopreserved PBMCs are suitable for Bmem analysis, facilitating logistical planning.
• These strategies enhance the practical application of Bmem characterization in large-scale studies.