Sample Handling and Submission

After collection, the samples should be submitted to the laboratory for analysis as soon as possible (ideally within 2 hours of sample collection). If there is a delay, in vitro changes (or pre-analytical errors) occur that altered the accuracy of the results. If the sample cannot be immediately submitted, their proper handling and storage can minimize these pre-analytical errors. For example, plasma or serum for chemistry analysis should be separated from blood cells immediately and as soon as clotting has occurred, respectively. The plasma or serum should be transferred to a clean tube without any additives. Because of normal metabolic cellular processes, delayed separation of plasma or serum causes a false decrease in glucose and pH. This will also interfere with analytes such as free ionized calcium (increase) and bicarbonate (decrease). Leakage of cellular constituents such as potassium may also occur in animals with high-potassium intra-erythrocytic concentrations (e.g. horses, cattle, and Asian dog breeds). Importantly, the use of gel separator tubes does not prevent these pre-analytical errors and the serum should be collected from the tube after centrifugation. In small animals, clotting typically occurs within a few minutes of placing the blood in a tube without anticoagulant. In large animal species, clotting make take up to 30 minutes. Clotting can be enhanced by incubating the samples at 37°C for at least 10 minutes. Once harvested, the plasma or serum should be stored at 4°C until submission (most analytes are stable for up to 48 hours if kept refrigerated).

Blood for a CBC is likewise affected by in vitro changes associated with delayed submission and sample aging. A commonly encountered pre-analytical error is that of in vitro cell swelling as cells begin to take up water from the plasma. This will typically manifest as a false increase in the red blood cell MCV and/or low MCHC (a pattern mimicking a regenerative anemia). Additionally, this will also cause a falsely increased hematocrit. Morphologic aging changes also typically occur. Red blood cells typically become echinocytic due to ATP depletion, this is important as it may obscure more significant red blood cell morphologies. Leukocytes, particularly neutrophils, also show cell swelling. Nuclear lobation becomes less distinct and Dohle bodies appear (Figure 1 A & B) mimicking a left shift and toxic change, respectively. Nuclei of lymphocytes become swollen, altering chromatin features, which may preclude accurate distinction between large reactive or neoplastic lymphocytes. With time, cells will rupture (Figure 1 C).

Segmented neutrophil, fresh blood smear.

Nuclear swelling and Dohle bodies (arrow), 48 h old blood

Ruptured (basket) cell consisting of free nuclear material

All these aging changes may alter the accuracy of the differential and the assessment of white and red blood cell morphology. Platelets may also start to clump, leading to falsely decreased counts or high MPVs. These pre-analytical errors can be minimized or circumvented by maintaining the sample cool by refrigeration or with an ice pack (avoiding direct contact as this can cause freezing and hemolysis of the sample) and making at least 1 blood smear immediately after sample collection. Blood smears should be submitted unfixed and unstained along with the anticoagulated blood. Note that unlike the anticoagulated blood, the blood smears should be store at room temperature (refrigeration causes cell lysis).

In conclusion, there are various and cumulative preanalytical variables that can affect the accuracy of laboratory analyses. It is important and necessary to be aware of these preanalytical variables, of how to prevent them or minimized them, and to take them into account when interpreting results.

1. CLSI GP41, Collection of Diagnostic Venous Blood Specimens, 7th Edition