Findings of recent studies indicate that flow cytometry (FCM) may be valuable in the diagnosis and prognostication of myelodysplastic syndromes (MDS). This approach appears particularly promising in patients with low-risk MDS without ringed sideroblasts and excess of blasts (i.e., with refractory anemia tout court) who have normal karyotype. These patients lack in fact any specific morphological or cytogenetic marker. However, the analytical methods reported so far require considerable technical skill, and therefore FCM has not yet become a routine procedure in the work-up of MDS patients. In this work, we developed a simple, reproducible FCM protocol for MDS and tested its validity prospectively. This study has been approved by the Ethics Committee, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy and by the Institutional Review Board of Nippon Medical School. The cytological diagnosis of MDS was made according to the WHO criteria by two independent cytologists who were blinded to clinical data. Three-color FCM was conducted at two laboratories (Tokyo and Pavia), which had received the details of the analytical method beforehand. The FCM protocol was developed in Tokyo and a part of which was reported previously (Leuk Res, 2008 32(5):699–707). The mandatory FCM parameters were CD34+ myeloblasts (% in all nucleated cells), CD34+ B-cell progenitors (% in all CD34+ cells), CD45 expression of CD34+ myeloblasts, and side scatter of mature myeloid cells. The optional parameters were CD11b, CD15, and CD56 expressions on CD34+ myeloblasts. These seven parameters were quantitatively analyzed and their reference ranges (RR) were determined using data from the cohort reported previously (

Blood. 2006; 108(3): 1037–44
). Bone marrow samples from 80 MDS patients with refractory anemia and normal karyotype, and from 82 controls were analyzed. Controls are patients who underwent routine diagnostic procedures for cytopenia and were eventually found to have conditions other than MDS and other clonal diseases. Abnormal data (outside the RR) in 2 or more parameters were common in MDS and were observed in 7 of 24 (29%) Japanese patients and 37 of 56 (66%) Italian patients when the four mandatory parameters alone were analyzed, and in 16 of 24 (67%) Japanese patients and 40 of 46 (87%) Italian patients when all seven parameters were analyzed (56 of 70 [80%] in total). A decreased CD34+ B-cell progenitor was the most common abnormality. By contrast, the occurrence of abnormalities in 2 or more FCM parameters was rare in control patients and was observed in 5 of 82 (6%) patients when all seven parameters were analyzed (56/70 versus 5/82, P < .0001). Therefore, when bone marrow samples lacking ringed sideroblasts and blast excess, and having normal karyotype show 2 or more abnormal FCM parameters, the likelihood ratio of MDS is 13.1 (95% confidence interval [CI], 6.4 to 29.3): the diagnostic sensitivity and specificity were 80% (95% CI, 74 to 84%) and 94% (95% CI, 89 to 97%), respectively. In conclusion, the findings of this study strongly indicate that the adopted FCM protocol is feasible and useful for diagnosing MDS in patients who lack specific morphological or cytogenetic markers.

Topics:
flow cytometry, karyotype determination procedure, myelodysplastic syndrome, bone marrow specimen, refractory anemias, antigens, cd15, cd45 antigens, cd56 antigens, cytopenia, macrophage-1 antigen

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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