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Evaluation of a novel mononuclear cell isolation procedure for serological HLA typing
P Schlenke1, H Klüter, M Müller-Steinhardt
1Institute of Immunology and Transfusion Medicine, University of Luebeck School of Medicine, D23538 Luebeck, Germany. schlenke@immu.mu-luebeck.de
Abstract:
Despite recent advances in DNA-based genotyping, the microcytotoxicity test is still broadly used for the determination of human leukocyte class I antigens in patients as well as organ donors and also for the detection of HLA antibodies. Excellent purity and viability of peripheral blood mononuclear cells (PBMC) are essential for reliable HLA typing results. Background staining and cell loss can contribute to impaired typing results or even cause misinterpretations. A novel isolation procedure using cell preparation tubes (CPT) with prefilled Ficoll was compared with the standard Ficoll gradient. We determined the recovery, purity, and viability of the PBMC after several periods of storage. Finally, the isolated cells were used for HLA class I typing, and background reactivities were scored. By using the CPT method, the recovery of PBMC was significantly higher than recovery with the standard technique (P
Insights
A new method using cell preparation tubes (CPT) improves peripheral blood mononuclear cell (PBMC) recovery and purity for human leukocyte antigen (HLA) typing. However, cell viability decreases over time with both CPT and standard methods, limiting HLA typing after 48 hours.
Area of Science:
- Immunogenetics
- Cellular immunology
- Transplantation immunology
Background:
- Microcytotoxicity testing remains crucial for human leukocyte antigen (HLA) typing and antibody detection.
- High purity and viability of peripheral blood mononuclear cells (PBMC) are critical for accurate HLA typing.
- Cell loss and background staining can compromise HLA typing results.
Purpose of the Study:
- To compare a novel cell preparation tube (CPT) method with standard Ficoll gradient for PBMC isolation.
- To evaluate PBMC recovery, purity, and viability after storage using both methods.
- To assess the impact of PBMC isolation methods on subsequent HLA class I typing and background reactivity.
Main Methods:
- Peripheral blood mononuclear cells (PBMC) were isolated using standard Ficoll gradient and a novel cell preparation tube (CPT) method.
- PBMC recovery, purity, and viability were assessed at various storage time points.
- Isolated cells were used for human leukocyte antigen (HLA) class I typing, and background reactivity was quantified.
Main Results:
- The CPT method yielded significantly higher PBMC recovery compared to the standard technique (P <= 0.001).
- PBMC purity remained stable with CPT, while granulocyte contamination increased over time with the standard method (P <= 0.001).
- Lymphocyte viability declined significantly over time with both methods, with CPT showing more dead cells; HLA typing was compromised after 48 hours due to high background.
Conclusions:
- The CPT method offers superior PBMC yield and purity for HLA typing compared to standard Ficoll gradients.
- Declining cell viability after 48 hours limits the utility of both isolation methods for time-sensitive applications like HLA typing and antibody screening.
- Optimized cell isolation protocols are essential for reliable immunogenetic testing.