Descriptive analysis of antigen expression pattern in refractory anemia, refractory anemia with ringed sideroblasts and refractory anemia with excess blast cases using flow cytometry

Abstract Abstract 595 Diagnosis and classification of myelodysplastic syndromes (MDS) is based on cytomorpholgy (CM) and cytogenetics (CG). By the identification of MDS-related aberrant antigen expression multiparameter flow cytometry (MFC) may add important diagnostic information. Examples include an aberrant expression pattern of CD13 and CD16 in granulocytes, an aberrante expression pattern of HLA-DR and CD11b in monocytes and the expression of lymphoid markers on myeloid blasts (Haematologica 2009:94:1124). To evaluate the potential role of MFC in the diagnostic setting of MDS we analyzed 1013 cases with suspected MDS by CM, CG, and MFC in parallel. Cases were classified by CM as refractory anemia (RA, n=31, 3.1%), refractory anemia with ring sideroblasts (RARS, n=27, 2.7%), refractory cytopenia with multilineage dysplasia (RCMD, n=64, 6.3%), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS, n=49, 4.8%), refractory anemia with excess of blasts 1 (RAEB-1, n=133, 13.1%), RAEB-2 (n=81, 8.0%), 5q- syndrome (n=24, 2.4%), chronic myelomonocytic leukemia (CMML, n=65, 6.4%), myelodysplastic syndrome unspecified (MDS-u, n=15, 1.5%), MDS borderline to acute myeloid leukemia (MDS/AML, n=6, 0.6%), MDS/myeloproliferative neplasia overlap (MDS/MPN, n=16, 1.6%), suspected MDS (n=225, 22.2%), reactive condition (n=266, 26.3%), and normal findings (n=11, 1.1%). Cytogenetic findings were normal karyotype (n=768, 75.8%), isolated deletion of long arm of chromosome 5 (del(5q), n=43, 4.2%), isolated aberrations of chromosome 7 (n=14, 1.4%), isolated trisomy 8 (n=30, 3.0%), isolated deletion of long arm of chromosome 20 (del(20q), n=21, 2.1%), complex karyotype (n=23, 2.3%), loss of Y-chromosome (n=43, 4.2%), other aberrations (n=71, 7.0%). Concordance between CM and MFC was 82.0% for diagnostic results in 788 cases with unequivocal CM. 277 of these 788 cases were classified by CM as not having MDS, 13 (4.7%) of which showed MDS-typical features by MFC. Additional 225 cases showed only minor dysplastic features by CM, 51 (22.7%) of which showed clear evidence of MDS by MFC. To further analyze the significance of MDS-related findings by MFC we then focused on cytogenetically aberrant cases without unequivocal MDS by CM. In 6/12 (50.0%) cases with no indication of MDS by CM and MDS-typical cytogenetic aberrations MFC revealed MDS characteristics. In another 11/23 (47.8%) cases with minor dysplastic features by CM and MDS-typical cytogenetic aberrations MFC revealed MDS characteristics. Furthermore, we compared blast counts as determined by CM and MFC and found a strong correlation (p<0.001) although the mean±SD percentage was higher as determined by CM as compared to MFC (4.67±4.18 vs. 3.78±2.97). Frequencies of aberrantly expressed antigens significantly differed between cases rated by CM as MDS (median number of aberrantly expressed antigens: 3), suspected MDS (1), and no MDS (0, p<0.001). In various cases MFC identified MDS-typical aberrant antigen expression in cell compartments not rated dysplastic by CM. Spearman rank correlation confirmed a highly significant relation between the number of aberrantly expressed antigens and IPSS (r=0.409, p<0.001). In 257 cases with data on overall survival (OS) the presence of MDS-related findings (≥3 aberrantly expressed antigens or a blast count >5% in MFC or a reduced side-scatter signal) resulted in significantly inferior 6-year-OS (68% vs. 100% p=0.008). The present analysis clearly demonstrates a diagnostic yield of MFC in addition to cytomorphology and cytogenetics in cases with suspected MDS. Disclosures: Kern: MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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Mitochondrial Ferritin Expression and Clonality of Hematopoiesis in Patients with Refractory Anemia with Ringed Sideroblasts.

Blood ◽

10.1182/blood.v106.11.3444.3444 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3444-3444 ◽

Cited By ~ 7

Author(s):

Matteo G. Della Porta ◽

Luca Malcovati ◽

Anna Galli ◽

Sabrina Boggi ◽

Erica Travaglino ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Early Stage ◽

Refractory Anemia ◽

Specific Marker ◽

Erythroid Cells ◽

Prognostic Information ◽

Sideroblastic Anemia ◽

Erythroid Progenitor ◽

Ringed Sideroblasts

Abstract Sideroblastic anemias are a heterogeneous group of disorders that have in common the presence of erythroblasts with iron-loaded mitochondria defined as ringed sideroblasts. We have previously demonstrated that mitochondrial iron deposition in these disorders is in the form of mitochondrial ferritin (MtF), suggesting that this latter may be a specific marker of sideroblastic anemia (Cazzola et al, Blood2003;101:1996–2000). The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts, which is generally associated with a relatively benign clinical course. In the present work, we studied the relationship between MtF expression and clonality of hematopoiesis in 55 consecutive female patients with low-risk MDS, including 20 cases with ringed sideroblasts and 35 cases without ringed sideroblasts. The expression of MtF, as well as that of cytosolic ferritin (H and L subunits) and of transferrin receptor (CD71), was evaluated by flow cytometry in bone marrow erythroid cells; in selected cases, these immunophenotypic investigations were also performed on liquid cultures of purified CD34-positive cells. X-chromosome inactivation patterns (XCIPs) were assessed in peripheral blood granulocytes and in bone marrow CD34-positive cells by analysis of both DNA methylation at the HUMARA and PGK loci and of IDS gene expression. Within informative females, 11 out of 12 patients with ringed sideroblasts displayed clonal XCIPs in granulocytes; by contrast, only 9 out of 22 patients without ringed sideroblasts displayed clonal XCIPs. Purified CD34-positive cells showed clonal XCIPs in 6 out of 7 patients with ringed sideroblasts but had polyclonal XCIPs in 4 out of 5 individuals without ringed sideroblasts. Flow cytometry evaluation of bone marrow erythroid cells showed that MtF expression was restricted to MDS patients with ringed sideroblasts. A close positive relationship was found between MtF and CD71 expression (r=.49, P=.001); this association may reflect the cytosolic iron deprivation induced by MtF overexpression. Analysis of cultured erythroid progenitor cells showed that MtF was detectable at a very early stage of differentiation from CD34-positive cells. Addition of erythropoietin to the culture system sustained the appearance of a polyclonal erythroid population in 2 out of 4 patients with ringed sideroblasts and clonal CD34-positive cells. These observations suggest that refractory anemia with ringed sideroblasts is a truly clonal stem cell disorder, while more than 50% of patients with refractory anemia without ringed sideroblasts have evidence of polyclonal hematopoiesis. The clonal pattern of CD34-positive cells and the early appearance of MtF during erythroid differentiation suggest that - despite be benign natural history of refractory anemia with ringed sideroblasts - the initial pathogenetic event in this condition occurs in multipotent stem cells. Although the mechanisms responsible for overexpression of MtF are still unclear, flow cytometry evaluation of this protein is a useful diagnostic tool that also provides helpful prognostic information.

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Diagnostic Utility of Flow Cytometry in Myelodysplastic Syndromes: A Prospective Validation Study in Low-Risk Patients with Normal Karyotype

Blood ◽

10.1182/blood.v112.11.3634.3634 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3634-3634

Author(s):

Kiyoyuki Ogata ◽

Matteo G. Della Porta ◽

Luca Malcovati ◽

Cristina Picone ◽

Norio Yokose ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

B Cell ◽

Myelodysplastic Syndromes ◽

Normal Karyotype ◽

Japanese Patients ◽

Low Risk ◽

Refractory Anemia ◽

Nippon Medical School ◽

Ringed Sideroblasts

Abstract 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.

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P088 Challenging diagnosis of refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS): detection of myeloid dysplasia by flow cytometry (FC)

Leukemia Research ◽

10.1016/s0145-2126(07)70158-3 ◽

2007 ◽

Vol 31 ◽

pp. S87-S88

Author(s):

P. Font ◽

D. Subira ◽

J. Loscertales ◽

C. Serrano ◽

S. Castanón ◽

...

Keyword(s):

Flow Cytometry ◽

Refractory Anemia ◽

Ringed Sideroblasts

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Aberrant Expression of CD Markers in Acute Myeloid Leukaemia

Haematology Journal of Bangladesh ◽

10.37545/haematoljbd201812 ◽

2018 ◽

Vol 2 (01) ◽

pp. 14-16

Author(s):

Abul Kalam Azad ◽

Md. Rafiquzzaman Khan ◽

ABM Hasan Habib ◽

Md. Abdul Wadud Miah ◽

Masuda Begum

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukaemia ◽

Myeloid Leukaemia ◽

Acute Leukaemia ◽

Antigen Expression ◽

Aberrant Expression ◽

Female Ratio ◽

Cd Antigen ◽

Cd Markers ◽

Acute Myeloid

Background: Aberrant expression of cluster differentiation (CD) antigen marker is associated with poor outcome of acute leukaemia. Objective: Aim of this study is to determine the frequency and pattern of aberrant expression of CD markers in acute myeloid leukaemia patients in Bangladesh. Methods: This retrospective data analysis was conducted in the Department of Haematology, Bangabandhu Sheikh Mujib Medical University (BSMMU) to assess the frequency of aberrant CD antigen expression in acute myeloid leukaemia from October 2016 to September 2017. During this period, we did one hundred flow cytometry of acute leukaemia patients and among them we found 48 acute myeloid Leukaemia (AML) who were included in this study. Result: Mean age of patients was 35 years (SD +14 years; Rang 3 to 50 years) with male: female ratio of 0.92. Four colour flow cytometry was done on fresh bone marrow aspirates and peripheral blood. Among 48 AML patients, aberrant CD expression was observed in 58% cases. CD5 and cCD79a lymphoid markers were seen to be expressed in 32% cases of AML. Aberrant cCD3 and CD7 were expressed in 29% and 25% cases respectively and aberrant CD10, CD19, cCD22 were expressed in 11%, 3%, 3% cases acute myeloid leukaemia patients respectively. Conclusion: Aberrant CD antigen expression is not uncommon in AML patients of Bangladeshi population that may adversely affect the treatment outcome of the disease.

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Increased Fraction of Circulating Activated Platelets in Acute and Previous Cerebrovascular Ischemia

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615191 ◽

1998 ◽

Vol 80 (08) ◽

pp. 298-301 ◽

Cited By ~ 62

Author(s):

Andreas Ruf ◽

Axel Vogt ◽

Christoph Lichy ◽

Florian Buggle ◽

Heinrich Patscheke ◽

...

Keyword(s):

Flow Cytometry ◽

Phase Contrast ◽

Matched Control ◽

Shape Change ◽

Antigen Expression ◽

Phase Contrast Microscopy ◽

Activated Platelets ◽

Contrast Microscopy ◽

Age And Sex

SummaryDetermination of circulating activated platelets may be helpful to estimate the prognosis and to stratify therapies in arterial vascular disorders including stroke. We used flow cytometry and phase contrast microscopy to study whether the fraction of platelets expressing p-selectin and CD63 and the fraction of platelets with shape change are increased in patients with acute and previous cerebrovascular ischemia.The proportion of platelets expressing activation dependent antigens was higher in patients with acute (n = 24; p-selectin: 8.23 ± 4.21%; CD63: 3.53 ± 2.53%) and with previous cerebrovascular ischemia (n = 46; 3.86 ± 1.98%; 2.80 ± 1.79%) as compared to age- and sex-matched control subjects (n = 35; 2.17 ± 0.96%; 1.79 ± 0.75%; p ≤0.005, respectively). In patients with previous ischemia, there was no difference between treatment with aspirin (n = 25) or phenprocoumon (n = 21). Hypertension, diabetes mellitus and smoking were not associated with increased antigen expression (analysis of variance). The fraction of discoid platelets and platelet counts were not significantly different between groups.Our results indicate increased expression of platelet neoantigens in acute and to a less degree in previous cerebrovascular ischemia. Ongoing platelet activation after cerebrovascular ischemia despite therapy with aspirin or phenprocoumon indicates that new anti-platelet drugs may be of benefit for these patients. Flow cytometry appears to be a useful tool to assess platelet function in cerebrovascular ischemia.

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