Stability of complete blood count parameters with storage: toward defined specifications for different diagnostic applications

In the pre-analytic phase, reliable specimen collection and transfer to the laboratory are fundamental to obtain high-quality test results in the hematology laboratory [1]. On behalf of the International Council for Standardization in Haematology (ICSH), several standards and guidelines for proper specimen collection, handling, and storage have been published [2–6]. Potassium and sodium salts of EDTA are commonly used anticoagulants for routine hematology determinations because they preserve the cellular components of blood [7]. As for the basic parameters of the complete blood cell count (CBC), earlier classical studies have reported acceptable stability after 24 h of storage of red blood cells (RBCs), white blood cells (WBCs) and platelets (PLTs), packed cell volume, hemoglobin concentration, mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), when EDTA is used at a concentration of less than 4 mg/mL of blood [2]. The pH difference induced by the use of different EDTA salts (K3, K2, and Na3) mainly affects the size of RBCs due to an osmotically dependent shrinkage, but the effect of dilution of the sample in automated analysis restores cells to their native shape, and no differences in mean cell volume (MCV) and microhematocrit are reported in samples analyzed between 1 and 4 h after collection. K2EDTA at a concentration of 1.5–2.2 mg of dipotassium EDTA dihydrate (K2EDTA.2H2O) per mL of blood is now considered as the anticoagulant of choice for modern automated CBC analyzers [6]. More recently, different authors have confirmed that the measurement of hemoglobin concentration and RBC count are stable up to 72 h after blood collection if blood is refrigerated at around 4 °C [8–11], while MCV and, consequently, the hematocrit value tends to increase after 6–12 h in a predictable measure [11, 12]. Time-related and concentration-related changes in PLT shape from discoid to spherical and swelling do occur in specimens collected in EDTA; as a result of these changes, the mean PLT volume (MPV) is not a fully reliable value and shows moderate interinstrument variability. In theory, the formation of giant PLTs can give rise to fragments that can result in a spuriously increased PLT count. However, the PLT count in specimens stored at 4 °C is considered stable for up to 24 or even 72 h [11], although some studies have shown a continuous mild decrease over time [13, 14]. The reticulocyte count, classically considered relatively unstable especially at room temperature [15] as a consequence of possible in vitro maturation to RBCs, has now been demonstrated as a robust parameter, stable for up to 72 h, in refrigerated samples, although some variations between different instruments have been described [9, 10, 16, 17]. WBC count with automated differential count is stable at 4 °C for at least 24 h, or even to 72 h, with significant differences depending on the type of automated blood cell analyzer [18, 19]: in particular, the monocyte count tends to increase, while eosinophil and lymphocyte counts tend to decrease over time; neutrophil count is stable up to 72 h with most lastgeneration instruments [12, 14, 19]. Irrespective of the type of anticoagulant, films made from blood stored not more than 1 h at room temperature (18–25 °C) are not easily distinguished from those smeared immediately after withdrawal. By 3 h at optimal EDTA concentration, minor changes may be observed, while by 12–18 h, morphological changes are striking at room temperature, so that refrigeration at 4 °C is necessary for long-term storage above 8 h [20]. Depending on the time the peripheral blood smear is prepared, as well as on EDTA concentration, morphological changes do occur in some but not all WBCs in the peripheral blood film [21]. Some neutrophils show nuclear swelling with changes in the chromatin, that is stained more homogenously compared to fresh nuclei, and loss of the structure of the lobes, that may become separated; the cytoplasmic rim may appear ragged or less well defined, and vacuolization and loss of granules may be observed. Nuclear shrinkage, chromatin condensation in dark masses, karyorrhexis, and degradation of cytoplasmic structure are early apoptotic changes [22]. Mononuclear cells are affected by similar morphological changes usually