Table 1.

Pitfalls in TCR clonality testing^a

Pitfall	Phenomenon	Solution/action
Bands/peaks just outside size range	Junctional regions/junctions outside 5–95% size range interval	Accept as true rearrangement product; in case of doubt, sequence for confirmation
Undersized bands/peaks	Internal deletion in, e.g., V segment	Potential rearrangement product; confirm by sequencing
Oversized bands/peaks	Extended amplification from downstream J	Potential rearrangement product; confirm by sequencing
Multiple clonal signals	Bi-allelic rearrangements; multiple rearrangements per allele or biclonality	Consider the number of potential rearrangements per allele and per locus and judge whether this fits with clonality or biclonality
Lack of clonal signal and lack of polyclonal Gaussian curve	Few T cells in sample	Check T cell presence by histology or flow cytometry
	Low DNA input/PCR	Check DNA concentration
	Poor DNA quality	Check DNA quality in control PCR
Selective amplification and pseudoclonality, due to low level of specific template	Few T cells in sample	Repeat PCR in multiplicates (same tissue, second independent DNA isolation, and/or related tissue) → compare patterns for consistency
Oligoclonal T-cell repertoire in PB of especially elderly individuals	Incomplete immune system, due to, e.g., immunosenescence	Repeat PCR in multiplicates (same tissue, second independent DNA isolation, and/or related tissue) → compare patterns for consistency and compare with primary process
Oligo-/monoclonality in histologically reactive lesion	Exaggerated immune response with dominant specificity	Repeat PCR in multiplicates (same tissue, second independent DNA isolation, and/or related tissue) → compare patterns for consistency
		(Re)evaluate histopathology → note that large germinal centers in the tissue may contain dominant T-cell clone(s)

^aAdapted from Langerak et al. [25]