The dilution factor of 5.4 results from the dilutions occurring during sample preparation steps A & B.
In Step A, 0.5 mL of the sample is added to a final volume of 1.5 mL, resulting in a 3 fold dilution of the sample.
In Step B, 1 mL of the sample from Step A is combined with a final volume of 1.8 mL, leading to a 1.8 fold dilution.
Consequently, the total dilution that the sample has experienced through these preparation/pre-treatment steps is 3 x 1.8 = 5.4 fold total dilution.
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