Separating capillary electrophoresis artifacts from true alleles
A single-source bloodstain is amplified with a 24-locus STR kit and injected on a 3500-series capillary instrument at 15 s and 1.6 kV. The analyst observes the following. (a) A green-channel peak of 1,200 RFU appears at the same scan number as a 2,400 RFU blue-channel D8S1179 allele. (b) A single-color spike of about 1,050 RFU appears at the same scan number in three capillaries of the same injection set. (c) A peak at the vWA locus is designated off-ladder at 9.3, with a height of 180 RFU; 9.3 is not present in the allelic ladder. (d) An expected D21S11 allele is 60 RFU. The internal size standard shows no off-ladder peaks, and the run's size-standard quality value is 0.98. The laboratory's analytical threshold is 125 RFU and its interpretation threshold is 200 RFU.
1. Evaluate observations (a) through (d) and state which peaks should be reported as alleles.
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(a) This is spectral pull-up, not an allele. A peak in a second dye channel at exactly the scan number of a strong peak, at roughly half its height, is the classic signature of inadequate color separation. It should be corrected by re-injecting at a lower injection time or with a fresh spectral calibration, not reported.
(b) A spike of the same height at the same scan number across several capillaries is an electrical or injection artifact. It is not sample-derived and is not reported. Check whether it overlaps any true allele; if it does, re-inject.
(c) The vWA 9.3 peak is above the analytical threshold, so it is real data, but an off-ladder designation is not the same as a confirmed allele call. It should be reported as off-ladder pending confirmation, not silently renamed.
(d) The 60 RFU peak is below the analytical threshold and must not be reported as an allele. Critically, the locus must not be reported as a homozygous genotype on the basis of the other allele alone; the correct result is a partial or inconclusive call at that locus.
2. What confirmatory steps are required before the vWA 9.3 peak can be reported?
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First, confirm reproducibility: re-inject the same amplification, and if possible re-amplify the extract, and verify that the 9.3 peak recurs at a consistent size and in proportion to the other vWA allele.
Second, verify sizing against the allelic ladder run in the same injection set, checking that the ladder itself shows no anomalous spacing at vWA and that the internal size standard is clean.
Third, inspect the peak for artifact characteristics: a one-base offset that is really a sizing or mobility artifact, a shoulder, or a dye blob. A width or shape inconsistent with neighboring alleles argues against a true microvariant.
Fourth, if the peak persists, confirm with a second kit or by sequencing the fragment. Only then should it be reported, and it should be reported as an off-ladder allele with its detected size and the confirmation performed. A single observation in one injection is not sufficient.
3. What should the report say about the D21S11 locus, and why does the distinction between analytical and interpretation thresholds matter here?
Read the worked answer
Report D21S11 as a partial result: one allele detected, with the second allele below the analytical threshold and therefore not interpretable. Do not report a homozygote.
The two thresholds do different jobs. The analytical threshold (125 RFU) is the level below which peaks cannot be reliably distinguished from noise, so a 60 RFU peak cannot be called. The interpretation threshold (200 RFU) governs whether a called allele is used in comparisons and statistics. A peak between the two would be a real but weak signal; a peak below both is simply not data for comparison purposes.
The practical consequence is that the locus is uninformative rather than homozygous. Reporting it as homozygous would improperly increase the apparent rarity of the profile and could create a false exclusion or a misleading statistic if the true second allele dropped out.