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

Abstract

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 K₃-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 K₃-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 K₃-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 K₃-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.

Keywords: flow cytometry; myelodysplastic syndromes; pre-analytical error.

Figure 1

Alterations of CD11b expression on peripheral blood granulocytes caused by delayed sample handling (N = 8). A and B represent samples collected in Na-heparin, C and D represents samples collected in K₃-EDTA. A and C represent FITC-labelled CD11b, while B and D represent PE-labelled CD11b. The groups were compared by Friedman test. Dunn’s multiple comparison test was applied as post hoc test.

Figure 2

Alterations of CD11b expression on peripheral blood monocytes caused by delayed sample handling (N = 8). A and B represent samples collected in Na-heparin, C and D represent samples collected in K₃-EDTA. A and C represent FITC-labelled CD11b, while B and D represent PE-labelled CD11b. The groups were compared by Friedman test. Dunn’s multiple comparison test was applied as post hoc test.