La scelta di differenti cloni e fluorocromi influenza la sensibilità diagnostica? Analisi di dati VEQ di UKNEQAS

Pathologists utilizing FACS for diagnostic pourposes in hematooncology find a wide selection of reagents which make sometimes difficult to choose between different clones and fluorochromes. In the last few years complex multiparameter analyses were made possible due to technological advancements such as the production of cheaper solid state laser sources and a wider panel of fluorochromes. Modern cytometers are able to detect at the same time many different wavelengths of fluorescent emission, and a monoclonal antibody of a given clone could be conjugated with several diverse flourochromes. Moreover, for a given CD manufacturers usually produce distinct clones that may behave in different way in vitro. This broad offer of clones and fluorochromes make possible to test with great precision several antigens at the same time, giving the chance to perform complex analysis such as the characterization of tiny cellular population in the peripheral blood and in the bone marrow. On the other hand diverse results for the same antigen analyzed in different laboratories or in the same laboratory at different times may be conditioned by the choice of the reagent. This pitfall is usually underestimated in everyday practice. With the precious collaboration of Liam Whitby from UKNEQAS we have been able to analyze data concerning 12 EQA exercises from the Leukemia Immunophenotipic Programme. In particular we examined 22 antigens, 14 diverse and 8 replicated in different EQA exercises. We calculated the percentage of laboratories reporting a positive result for a given antigen according to the British Committee for Standards in Haematology (BCSH) guidelines which set a threshold of positivity at 20 % of the cells for acute leukemias and at 30 % of the cells for chronic leukemias. With regard to fluorochromes we found a better performance of the PE conjugate for eight antigens (CD1a, CD4, CD7, CD10, CD22, CD23, CD23, CD34 and CD79B) and of the FITC conjugate for the TdT, while no differences between FITC and PE reagents were noticed for CD13, CD20, CD33 and MPO. Concerning the clones we found out that for a given CD diverse reagents show a different behaviour. In particular TdT Dako, CD20 Becton Dickinson (BD), CD1a Dako, CD4 BD, CD22 BD and CD79b Beckman Coulter (BC) showed a better performance compared to the same products from other manufacturers. These data show that reagents do not behave in the same way despite a common CD clusterization and that their use without taking into account these differences may bring to misleading diagnosis.