Background: Although it is recommended that primary tubes containing an additive should be mixed several times, there is no evidence that unmixed specimens will provide unreliable results in hematological testing. Methods: Three primary 3.0-mL siliconized vacuum tubes containing 5.4 mg K2 ethylene diamine tetraacetic acid (EDTA) were sequentially collected from 20 healthy volunteers. The first was not inverted, was left standing in a vertical position, and then analyzed. The second and third tubes were respectively inverted 6 and 12 times immediately after collection and then analyzed. Results: When compared with the reference specimens inverted 6 times, results on unmixed specimens revealed significant decreases for red blood cell count, hemoglobin, hematocrit and platelets count, whereas the mean platelet volume was significantly increased. In none of the specimens were results flagged for platelet clumping, nor did the differences exceed the acceptable limits of bias. Conclusion: Provided the tubes are filled to their nominal volume, the blood-to-anticoagulant mixture occurring during sample collection may be adequate.