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Updated: May 24, 2026
DNA-barcode-based Multiplex Immunofluorescence Imaging to Analyze FFPE Specimens from Genetically Reprogrammed Murine Melanoma
Published on: June 6, 2025
Comparison of formalin-fixed and snap-frozen samples analyzed by multiplex ligation-dependent probe amplification for
Sarah L Lake1, Helen Kalirai, Justyna Dopierala
1Department of Molecular & Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom.
Purpose:
Survival of patients with uveal melanoma (UM) correlates strongly with chromosomal abnormalities, such as monosomy 3 and polysomy 8q. Studies have shown multiplex ligation-dependent probe amplification (MLPA) to detect reliably chromosomal aberrations in UM using frozen samples. To date, the concordance between MLPA data generated from frozen and fixed samples has not been investigated. Our study aimed to compare MLPA data obtained from fixed and frozen tissues to determine whether fixed samples can be used in UM genetic prognostic testing.
Methods:
Adjacent samples were taken from 20 UM cases, with one sample being snap-frozen, and the second being formalin-fixed and paraffin-embedded (FFPE). MLPA analysis was performed using the P027.B1 assay. The chromosome arm copy numbers obtained for each pair of UM samples then were compared.
Results:
Of the 20 UM cases 18 gave MLPA results from FFPE material that passed quality control thresholds. When chromosome 3 copy number was classified successfully (loss, disomy or gain) using FFPE material, the corresponding frozen sample showed concordance in 100% of cases. However, even when FFPE samples passed quality thresholds, chromosome arm copy numbers for 3p and 3q were "unclassifiable" in 28% and 11% of cases, respectively. This compared to "unclassifiable" cases in 0% and 6% of the frozen UM samples.
Conclusions:
Whenever possible, fresh or snap-frozen tissue should be used for UM genetic prognostic testing by MLPA. When only fixed tissue is available, MLPA can be used to determine reliably chromosome 3 copy number. However, some tumors will be unclassifiable where DNA quality is poor.
