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Table 1 Specimen requirements and assay specifications of KRAS genotyping by laboratories

From: Reliability of KRAS mutation testing in metastatic colorectal cancer patients across five laboratories

  Lab #1 (Sequencing) Lab #2 (Sequencing) Lab #3 (Sequencing) Lab #4 (Primer Extension) Lab #5 (Real Time PCR)
Specimen Requirements Preferred sample type*:
Slides from FFPE block
1  H&E stained slide sections with tumor circled; 4 matching unstained slides,
10 microns each.
Preferred sample type: Archival FFPE or frozen surgical biopsies confirmed to contain >50% tumor by a surgical pathologist.
1 H&E slide;
5 unstained sections,
10 microns each.
Preferred sample type: FFPE tissue
6 unstained sections,
10 microns each.
Preferred sample type: Pre-cut slides from FFPE. Send all slides within 5–7 days of cutting. Air dry. Do not oven dry. Store specimen at room temperature (20–23.5°C).
5 unstained sections,
7 microns each
Preferred sample type: FFPE block, unstained slides, or fresh snap frozen biopsy
5 unstained sections,
7 microns each
Genotyping Method: PCR amplification followed
by Direct Sanger sequencing
(Big Dye v. 1.1)
Detected mutations: KRAS codons 12 and 13
Method: PCR amplification followed by standard bidirectional sequencing on ABI 3100.
Detected mutations: KRAS codons 12 and 13
Method: PCR amplification followed by sequencing.
Detected mutations: KRAS codons 12,
13 and 61
Method: Single nucleotide primer extension with fragment analysis by capillary electrophoresis using a modified SNaPshot assay.
Detected mutations:
KRAS codons 12 and 13
Methods are propietary: qualitative real time PCR
Detected mutations: KRAS codons 12 and 13
Lower Limit of Detection 20% when ≥ 40% tumor cells present 20% 15-20% 10% when ≥ 2% tumor cells present 1-5%
  1. *For this study, slides prepared from Formalin-fixed paraffin embedded (FFPE) blocks were sent to each lab.