The impact of delayed sample handling and type of anticoagulant on the interpretation of dysplastic signs detected by flow cytometry

INTRODUCTION: A growing body of evidence supports the usefulness of dysplastic signs detected by flow cytometry in the diagnosis of myelodysplastic syndromes (MDS). Our aim was to assess the impact of pre-analytical variables (delayed sample handling, type of anticoagulant, and different clones of antibody) in the interpretation of flow cytometric results. MATERIAL AND METHODS: Bone marrow samples were labelled and analysed immediately after aspiration and on two consecutive days. The effect of anticoagulant type was evaluated in 16 bone marrow samples. Thirty-seven different immunophenotypic variables were recorded after eight-colour staining. Furthermore, 8 normal peripheral blood samples collected in K(3)-EDTA and Na-heparin were examined with different clones of CD11b antibodies and four parameters were recorded with both anticoagulants on two consecutive days. RESULTS: Fourteen significant differences were detected in the initial immunophenotype of fresh samples collected in K(3)-EDTA and Na-heparin. Regardless of the anticoagulant type, eleven parameters remained stable despite delayed sample handling. Due to delayed sample processing, more alterations were detected in the samples collected in K(3)-EDTA than in the samples collected in Na-heparin. The type of CD11b clone influenced the reduction of fluorescence intensity only in samples collected in K(3)-EDTA, where the alterations were contrary to the changes observed in Na-heparin. CONCLUSIONS: Delayed sample processing causes considerable immunohenotypic alterations, which can lead to false interpretation of the results. If delayed sample evaluation is unavoidable, markers that remain more stable over time should be considered with more weight in the diagnosis of MDS.