The complete blood count (CBC) is one of the most frequently performed analytic tests in laboratory medicine. Flow cytometry is used to distinguish and count blood cell populations. According to the Medical Devices Act, participation in round robin tests for the complete blood count has been mandatory since 2002. In this way, external quality assurance to ensure the comparability of the measurement results – and, thus, patients' safety – has been established. For the evaluation of such ring trials, medical professional associations use reference measurement values from PTB as target values.
Due to the complexity of biological systems, it is crucial for many analytes to meet the requirements of a primary reference measurement procedure, i. e. for traceability to the SI units, a complete analysis of the influence quantities and interference factors as well as a detailed consideration of the measurement uncertainties. Hence, also for blood cell counting, standardized procedures potentially suited as primary reference measurement procedures are available only for few types of cells, namely erythrocytes, leukocytes and platelets. For erythrocytes, the measurement procedure developed at PTB complies with the requirements of a primary reference measurement procedure.
The measurement procedure is based on the flow-cytometric detection of cells by impedance measurements using a novel measuring sensor. It is characterized by applying a front sheath flow for hydrodynamic focussing of the blood corpuscles and a rear sheath flow. The latter prevents particles which have passed the orifice from re-circulating and, thus, prevents counting errors. Counting losses, which are due to random coincidences and depend on instrumental parameters and the properties of the sample, are taken into account by an examination of serial dilutions. The analysis of dilution series allows the determination of the reference measurement value of the erythrocyte concentration by extrapolation to vanishing volume fractions of the sample in the measurement suspension. The application of this method results in measurement uncertainties of less than 0.75 % for red blood cells being one order of magnitude below the assessment limits valid for passing the round robin tests.
The researchers' next goal is to develop primary reference measurement procedures for other types of cells such as CD4-positive T-helper lymphocytes. Concentrations of these cells lying below a certain threshold level are indicative of an HIV infection. In addition, investigations are carried out to develop and validate a secondary reference measurement procedure based on relative concentration measurements. Such a secondary procedure would be applicable in routine laboratories and would allow end users to verify the quality of their own analyses. In the long run, PTB supports the introduction of internationally harmonized standards in order to ensure the cross-border comparability of medical diagnosis.
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