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Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation
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HLA class I sequence-based typing using DNA recovered from frozen plasma.

Laura A Cotton1, Manal Abdur Rahman, Carmond Ng

  • 1Faculty of Health Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.

Journal of Immunological Methods
|May 16, 2012
PubMed
Summary

We developed a fast, affordable method for HLA class I typing using frozen plasma DNA. This approach achieves 100% concordance with cell-based methods, offering a reliable alternative when samples are limited.

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Area of Science:

  • Immunogenetics
  • Molecular Biology
  • Genomic Medicine

Background:

  • Human Leukocyte Antigen (HLA) typing is crucial for transplantation and disease association studies.
  • Traditional HLA typing methods often require large amounts of genomic DNA from peripheral blood mononuclear cells (PBMC).
  • Limited availability of PBMC or DNA can hinder HLA typing, especially with older or challenging samples.

Purpose of the Study:

  • To establish a rapid, reliable, and cost-effective method for intermediate-to-high-resolution sequence-based HLA class I typing.
  • To evaluate the feasibility of using frozen plasma as a source of genomic DNA for HLA typing.
  • To compare the accuracy and concordance of HLA typing results derived from plasma versus PBMC.

Main Methods:

  • Nucleic acids were isolated from matched frozen PBMC and plasma samples using commercial kits.

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  • Specific regions of HLA-A, -B, and -C genes (exons 2 and 3) were amplified using nested PCR.
  • Direct sequencing and subsequent chromatogram analysis were performed, with allele interpretation using a web-based tool.

    • Main Results:

    • High-quality nucleic acid yields were obtained from both PBMC and plasma.
    • Amplification rates for HLA-A, -B, and -C were 100% from both DNA sources.
    • 100% concordance was observed between PBMC and plasma-derived HLA types at allele and protein levels, with >99.9% nucleotide concordance.

    Conclusions:

    • Frozen plasma is a viable and effective source for genomic DNA for sequence-based HLA class I typing.
    • This method offers comparable specificity to conventional approaches and is suitable for situations with limited cell samples or DNA quantities.
    • The protocol is rapid, reliable, cost-effective, and adaptable for various sample types and ages.