Table 5.
Quality Control/Quality Assurance for Single Cell PCR
| Process | Measures |
|---|---|
| Routine/general QC | Comprehensive training/protocols (esp. contamination control) |
| Avoid specimen mix-up (multiple samples/embryos per patient) | |
| Overlap batches of tested and untested reagents | |
| Test all reagents prior to a clinical case | |
| Check temperatures of water-baths/thermal cyclers, etc | |
| Pipette calibration | |
| External quality assessment (unavailable at present) | |
| Ensure appropriate testing | Medical genetics consultation recommended |
| Verification of diagnosis (documentation or laboratory re-test) | |
| Apply PGD test offered to DNA/single cells from the couple | |
| Karyotype couple to exclude chromosomal abnormality? | |
| Assay development | Minimum number of single cells analyzed for assay development |
| Use heterozygous single cells to establish ADO/amplification rates | |
| Blastomere analysis for assay development | |
| Analyze DNA from−/−,+/− and +/+ sources | |
| Optimize primer design (particularly first round of nested PCR) | |
| Perform “dummy-runs” in simulated case conditions | |
| Clinical assay | Selection of mononucleate blastomeres only for analysis |
| Observe cell transfer to reaction tube | |
| Use of check gel to avoid post-PCR processing of failed samples | |
| Use of positive and multiple negative controls per clinical assay | |
| Contamination (observe precautions described in Table 3 ) | |
| Allele dropout (observe precautions described in Table 4 ) | |
| Minimize turn-around-time (for timely embryo selection/transfer) | |
| Mutation detection | Design PCR such that normal allele is cut into new product sizes |
| Use of internal controls for restriction enzyme digestion | |
| Purify PCR product prior to restriction digestion (if necessary) | |
| Establish cut-off values for failed amplifications/contamination in fluorescent PCR | |
| Sequence using forward and reverse primers | |
| Documentation | Labeling± color coding of tubes |
| Worksheet to contain all tube labels, gel loading sequence, etc. | |
| Diagnostic laboratory supervisor/director sign off for all cases | |
| Witness in IVF laboratory for embryo selection and transfer | |
| Misdiagnosis rates/ADO rates | Assess single blastomeres from non-transferred embryos |
| Confirm PGD result by amniocentesis/CVS/cord blood |