Cell-free DNA fragmentation patterns in amniotic fluid identify genetic abnormalities and changes due to storage.
Author(s): Peter I, Tighiouart H, Lapaire O, Johnson KL, Bianchi DW, Terrin N
Publication: Diagn Mol Pathol, 2008, Vol. 17, Page 185-90
PubMed ID: 18382362 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of storage duration and chromosomal number on the cell-free DNA fragmentation pattern in amniotic fluid (AF).
Conclusion of Paper
When euploid AF was stored frozen rather than analyzed fresh the retention factor (RF) curves shifted toward one reflecting a loss of large DNA pieces and an increase in smaller fragments. Interestingly, the interindividual variation in RF curves among euploid specimens was small, regardless of storage duration, but none of the 10 aneuploid specimens stored for 1-6 months had a comparable RF curve (banding pattern) to euploid specimens stored for 4-6 months.
Studies
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Study Purpose
The purpose of this study was to determine the effects of storage duration and chromosomal number on the cell-free DNA fragmentation pattern in amniotic fluid (AF). Supernatants of leftover AF were obtained from pregnant women (15-34 weeks) carrying fetuses with Turner syndrome (4), triploidy (4), trisomy 13 (3), trisomy 18 (8), trisomy 21 (10) or normal chromosome number (36). Ten specimens were analyzed fresh, while the remainder were stored at -80°C for 1-79 months before extraction. DNA was extracted from specimens using the QIAamp DNA maxi kit and DNA yield was determined by the band pattern of real-time PCR amplicons of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Summary of Findings:
When euploid AF was frozen instead of analyzed fresh or with increasing storage duration (4-79 months) of euploid AF, retention factor (RF) curves shifted toward one reflecting a loss of large DNA pieces (low RF) and an increase in smaller fragments. The size of the largest fragment (the first RF peak location) was comparable in euploid AF analyzed fresh and that stored for 11-12 months, but was smaller when stored 17-20 months and absent when stored 59-79 months. However, the height of the peak (the distance between maximum and minimum) decreased linearly during the first 12 months of euploid AF storage (r2=0.83, p<0.0001), after which it could not be calculated due to peak loss. Peaks representing the smallest DNA fragments were comparable in euploid AF analyzed fresh and those archived 59-79 months, but was not observed in euploid AF specimens stored for 4-6 months, 11-12 months or 17-20 months. Interestingly, interindividual variation in the RF curves among euploid specimens was very small, regardless of storage duration. Of the 10 aneuploid specimens stored for 1-6 months, none had a comparable banding pattern to that observed in the 8 euploid specimens stored for 4-6 months, with differences being due to location of first peak (largest DNA fragment size, 7 of 10 specimens) and first minimum in the curve(smallest size of that fragment, 8 of 10 specimens), and the percent change between these factors (9 of 10 specimens).
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Down Syndrome
- Pregnant
Platform:
Analyte Technology Platform DNA Electrophoresis DNA Real-time qPCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 0 months
4-6 months
11-12 months
17-20 months
59-79 months
1-6 months
Real-time qPCR Specific Targeted nucleic acid GAPDH
Preaquisition Diagnosis/ patient condition Pregnant with euploid fetus
Pregnant with aneuploid fetus
Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)