Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study

Abstract

Background: The diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts.

Design and methods: We designed a flow cytometry protocol applicable in many laboratories and verified its diagnostic utility in patients without those diagnostic markers. The cardinal parameters, analyzable from one cell aliquot, were myeloblasts (%), B-cell progenitors (%), myeloblast CD45 expression, and channel number of side scatter where the maximum number of granulocytes occurs. The adjunctive parameters were CD11b, CD15, and CD56 expression (%) on myeloblasts. Marrow samples from 106 control patients with cytopenia and 134 low-grade myelodysplastic syndromes patients, including 81 lacking both ringed sideroblasts and cytogenetic aberrations, were prospectively analyzed in Japan and Italy.

Results: Data outside the predetermined reference range in 2 or more parameters (multiple abnormalities) were common in myelodysplastic syndromes patients. In those lacking ringed sideroblasts and cytogenetic aberrations, multiple abnormalities were observed in 8/26 Japanese (30.8%) and 37/55 Italians (67.3%) when the cardinal parameters alone were considered, and in 17/26 Japanese (65.4%) and 42/47 Italians (89.4%) when all parameters were taken into account. Multiple abnormalities were rare in controls. When data from all parameters were used, the diagnostic sensitivities were 65% and 89%, specificities were 98% and 90%, and likelihood ratios were 28.1 and 8.5 for the Japanese and Italian cohorts, respectively.

Conclusions: This protocol can be used in the diagnostic work-up of low-grade myelodysplastic syndromes patients who lack specific diagnostic markers, although further improvement in diagnostic power is desirable.

Figure 1.

Analysis of four cardinal parameters from a single cell aliquot stained with CD10-FITC, CD34-PE, and CD45-PerCP antibodies. (A) All nucleated cells (R1) and cells with relatively low SSC (R2). (B) Cells in R2 in panel A were displayed on a CD34-versus-CD45 plot. CD34⁺ cells with intermediate CD45 expression were gated (R3). (C) Cells in R3 in panel B were displayed on a CD45-versus-SSC plot. A cluster of CD34⁺ B-cell progenitors was identified in the lower left region of CD34⁺ cells (R5, arrow). The reliability of the R5 region was confirmed based on CD10 positivity. Cells in R4 were composed mainly of myeloblasts (Mbls) and thus simply called CD34⁺ Mbls in this paper. (D) Granulocytic cells other than Mbls (R6, called granulocytic cells in this paper) and lymphocytes (R7) were gated on a CD45-versus-SSC plot. (E) Cells in R6 in panel D were displayed, and the CD10⁻ fraction was gated (R8). (F) SSC of CD10⁻ granulocytic cells (upper panel) and lymphocytes (lower panel). SSC peak channel values (SSC channel number where the maximum number of cells occurs) of both fractions were computed using the software. (G) CD45 expression of CD34⁺ Mbls (upper panel) and lymphocytes (lower panel). Mean fluorescence intensity (MFI, GeoMean) of CD45 of both fractions was computed.

Figure 2.

Analysis of seven flow cytometry parameters in the prospective cohorts. The horizontal lines in each boxplot represent the 90^th, 75^th, 50^th, 25^th, and 10^th percentiles. Circles are outliers. Controls are nonclonal cytopenic patients. “All” indicates all low-grade myelodysplastic syndromes (MDS) patients, “With” indicates low-grade MDS with conventional markers, and “W/O” indicates low-grade MDS without conventional markers. Shaded areas are the predetermined reference ranges.