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The online version of this article contains a supplementary appendix. BackgroundThe 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 MethodsWe 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. ResultsData 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. ConclusionsThis 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.Key words: myelodysplastic syndromes, flow cytometry, diagnosis.Citation: Ogata K, Della Porta MG, Malcovati L, Picone C, Yokose N, Matsuda A, Yamashita T, Tamura H, Tsukada J, and Dan K. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study. Haematologica 2009;94:1066-1074. doi:10.3324/haematol.2009 This is an open-access paper. Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study

were analyzed using 4-color flow cytometry (FCM). We objectively determined reference ranges of 13 parameters related to CD34 ؉ cells with data from controls. InLGw/oRS patients, various abnormalities of CD34 ؉ cells-eg, decrease in CD34 ؉ B-cell precursors, aberrant expression or overexpression of various antigens on CD34 ؉ myeloblasts-were observed. We constructed a reproducible, flow cytometric scoring system for LGw/oRS diagnosis. High scores were observed in 16 of 27LGw/oRS patients, regardless of the presence or absence of chromosomal aberrations, but not in any of the 90 controls. Among LGw/oRS patients with chromosomal aberrations, patients with trisomy 8 or del20(q) had low FCM scores (P ‫؍‬ .002). As a result, most LGw/oRS patients were identified based on high FCM score, chromosomal aberration, or both.

During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-␥ (IFN␥) and tumor necrosis factor-␣ (TNF␣) on MDS blasts. This induction was associated with the activation of nuclear factor-B (NF-B) and nearly completely blocked by an NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1 ؉ MDS blasts had greater intrinsic proliferative capacity than B7-H1 ؊ MDS blasts when examined in various assays. Furthermore, B7-H1 ؉ blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken IntroductionB7-H1 (CD274), which was identified by us as a costimulatory molecule, plays a crucial role in T-cell regulation in various immune responses. 1,2 B7-H1 molecules deliver a costimulatory signal through an unknown receptor on naive T cells. [1][2][3] They also deliver an inhibitory signal to activated T cells through programmed cell death 1 (PD-1) molecules, 4 which are a type I transmembrane protein belonging to the CD28 receptor family and were originally identified in T cells undergoing apoptosis. 5 B7-H1 expression is detected not only on antigenpresenting cells but also on activated T cells and some tumor cells (ie, renal cell, colon, breast, and lung carcinoma, and Hodgkin lymphoma). [6][7][8][9][10] Rodent data suggest that B7-H1 molecules on tumor cells deliver negative signals through PD-1 and other receptors on tumorspecific cytotoxic T lymphocytes and inhibit antitumor immune responses. 11,12 Consistent with those data, it was reported that in patients with renal cell carcinoma and breast cancer, patients whose tumor cells expressed B7-H1 had a poor prognosis. 9,13 In a mouse leukemia model in which mice were immunized with irradiated DA1-3b leukemia cells and then challenged with live DA1-3b cells, only leukemia cells expressing high levels of B7-H1 survived for a long period. Moreover, these cells gained tolerance to specific cytotoxic T lymphocytemediated killing. 14 Therefore, B7-H1 molecules on leukemia cells may be associated with immune evasion in this model.Myelodysplastic syndromes (MDS) are clonal hematologic stem cell disorders characterized by cytopenias, excessive apoptosis of hematopoietic cells, and a high risk of progression to acute myeloid leukemia (AML). In MDS, various immune abnormalities, including lymphopenia and T-cell dysfunction, have been reported, 15-17 although data on B7-related molecules, in particular B7-H1, are lacking. With disease progression, that is, with i...

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Idiopathic thrombocytopenic purpura (ITP) occurs more commonly in young women during the reproductive years. To obtain information for management of ITP in pregnancy, we performed a nationwide retrospective survey. Findings from a total of 284 pregnant women with ITP and their 286 newborn infants were available for analysis. The bleeding tendency at delivery was managed chiefly with corticosteroid, intravenous high-dose gamma-globulin, and platelet transfusion. Maternal complications occurred in 77 cases (27.1%) and were frequently seen in cases with poor control of ITP. Neonatal abnormalities, which were not influenced by the clinical state of the mother, occurred at a frequency of 17.8%. Thrombocytopenia in neonates occurred in 48 cases (22.4%), and bleeding tendency was found in 16 cases (6.3%) without severe bleeding. Prediction of thrombocytopenia in neonates was difficult. However, infants from splenectomized mothers with well-controlled ITP showed thrombocytopenia more frequently than those from nonsplenectomized mothers. Mothers treated with steroids at doses greater than 15 mg/day showed a high frequency of maternal complications and fetal abnormal body weight. These observations will be useful in the management of pregnant women with ITP and their infants.

SUMMARYCongenital patients who lack natural killer (NK) cell activity experience repeated polymicrobial infections. NK cell activity varies significantly among normal people, but it is unknown whether this variation influences their ability to fight infections. This study examined this concern. NK cell activity and other variables, i.e. age, sex, performance status (PS), serum albumin value, lymphocyte and neutrophil counts, various lymphocyte subsets, etc. were determined for 108 immunologically normal elderly subjects who were in nursing homes due to an impaired PS. We analysed for correlations between these variables and the follow-up results of the subjects. Forty-eight subjects developed infection(s) during the first year of follow-up. A low NK cell activity was associated with the development of infection (P 0´0105, multivariate logistic regression analysis). The relative risk for the development of infection increased in accordance with the decrease in the NK cell activity. Eleven subjects died of infection during the study period. A low NK cell activity was associated with short survival due to infection (P 0´0056, multivariate Cox's proportional-hazards regression analysis). Our data indicate that low NK cell activity is associated with development of infections and death due to infection in immunologically normal elderly subjects with an impaired PS.

Purpose: The B7 family molecules have been shown to regulate immune responses in both positive and negative fashions.Their roles in the progression of human cancers, however, are not well established. The aim of this study was to examine whether leukemic cells of acute myeloid leukemia express functional B7 family molecules and, if so, whether such expression has any clinical significance. Experimental Design: The expression of four B7 family molecules, B7.1, B7.2, B7-H1, and B7-H2, on leukemic cells from acute myeloid leukemia patients was analyzed by flow cytometry. The function of the expressed molecules was examined by the allogeneic mixed lymphocyte-leukemic cell reaction, and their relationship to the clinical data and survival was analyzed. Results: Although B7.1and B7-H1expressions were rare, the cells from a substantial number of acute myeloid leukemia cases expressed B7.2 and B7-H2 molecules [mean percentages of B7.2-and B7-H2-positive cells were 28.9% (n = 58) and 15.3% (n = 59), respectively]. Patients in whom >25% of leukemic cells expressed B7-H2 had significantly shorter survival, and this B7-H2 positivity had the strongest prognostic value when B7-H2 and other prognostic factors were analyzed together by multivariate analysis (P = 0.0108). Furthermore, B7.2 expression was associated with hyperleukocytosis (P = 0.026). Consistent with this finding, acute myeloid leukemia cells expressing B7.2 and B7-H2 induced allogeneic CD4 + T cells to proliferate and secrete interleukin-4 and interleukin-10 in vitro, effects that were partially blocked by antibodies against B7.2 and B7-H2. Conclusions: Our results indicate the expression of functional B7.2 and B7-H2 molecules, and these molecules may facilitate progression of acute myeloid leukemia.