Jörg Neukammer, Manuela John, Andreas Kummrow, Stephan Reitz, Sandra Schädel, Karin Schulze, Klaus Witt
In support of medical diagnosis the concentration of different cells in body fluids has to be known with high accuracy. In the working group "Flow Cytometry and Microscopy" of the Physikalisch-Technische Bundesanstalt (PTB) reference procedures have been developed to determine reference values for the measurands of the Complete Blood Count (CBC). The reference values for the concentration of erythrocytes, platelets and leukocytes as well as the concentration of haemoglobin and the haematocrit value are provided by the PTB to evaluate round robin tests organised by German medical associations. In Germany, participation of clinical laboratories for external quality control is mandatory and regulated by the Guidelines of the German Medical Association (Richtlinien der Bundesärztekammer, RiLiBÄK). In addition, the maximal admissible deviation from target values determined by reference laboratories is defined in the RiLiBÄK.
In flow cytometry, blood cells are detected and differentiated by impedance changes, light scatter or fluorescence signals when passing the measuring sensor or interaction zone. Depending on the instrumental design, count rates between several 100 Hz up to 10 kHz can be obtained. To derive reference measurement values for the concentration of cells, the procedures developed at the PTB are based on gravimetrically controlled volume measurements in combination with serial dilutions to correct for coincidence loss as well as cell loss, e.g. due to adhesion. The method relies on the measurement of the cell concentration as function of the volume fraction of the (blood) sample in the measurement suspension and extrapolation to zero volume fraction. Studies on coincidence loss and correction by serial dilutions were accomplished by several author decades ago utilizing the Coulter principle, i.e. resistive pulse detection. This method of coincidence correction is not only suited for impedance cell counters but can also be applied for dead time correction in optically based instruments where cell differentiation is achieved by light scatter or fluorescence of immunologically stained cells.
It is important to point out that the counting loss due to the dead time not only depends on the instrument but also on the size and the rheological properties of the cells. Hence, calibration of the respective instrument is not sufficient and the determination of reference measurement values requires the preparation of a dilution series for each blood sample. The modus operandi to derive reference values of concentrations of erythrocytes and leukocytes is described in German standards and recommended by the International Council for Standardisation in Haematology (ICSH). The series of German standards concerning the complete blood count also includes reference procedures to determine the haemoglobin concentration in blood, blood collection and sample preparation, characteristic quantities for erythrocytes and the determination of the packed cell volume by centrifugation. In addition, a reference procedure to determine platelet concentrations is described, which includes specific immunological staining to identify the target cells.