The Challenge Of Quantifying CD34+ Myeloid Cells In Myelodysplastic Syndromes With Less Than 5% Bone Marrow Blasts. Reproducibility Among 6 Flow Cytometry Observers

Abstract Introduction In low-risk myelodysplastic syndromes (MDS), the morphological bone marrow (BM) blast cell count between 0 and 2%, and >2 - <5%, has demonstrated prognostic value and is critical for R-IPSS. Flow cytometry immunophenotyping (FCI) provides an accurate way for quantification of the immature BM cell compartment through the identification of CD34+ cells and may contribute to a better characterization of these MDS low-risk categories. However, there is a wide variety of FCI strategies to study the CD34+ cells, without a universal consensus on how many and which markers should be used to reach their best identification. There are some studies evaluating the correlation between FCI and the morphological blast cells count, but it is not clear if FCI allows a good concordance among several observers when the morphological blast count is <5%. Objectives 1-To explore the concordance among 6 FCI observers to quantify the CD34+ myeloid BM cells from patients diagnosed with MDS with <5% BM blasts 2- To study the correlation between FCI and morphology for detecting <5% BM blast cells. 3- To determine if the mophological threshold of 2% is reproducible by FCI. Methods FCI data files from 48 MDS BM samples with <5% blasts were simultaneously and independently evaluated by 6 FCI observers from 6 different Spanish hospitals. According to the WHO criteria patients were distributed as follows: 3 refractory cytopenia with unilineage dysplasia; 13 refractory anemia with ring sideroblasts; 25 refractory cytopenia with multilineage dysplasia; 1 unclassifiable MDS; 2 chronic myelomonocytic leukemia and 4 therapy-related myeloid neoplasms. Each participant contributed with 8 samples and all files were exchanged among them. All of them used the INFINICYTTM software program for analysis according to their usual strategies. The morphological quantification of BM blast cells was provided by each centre and was blinded to the others. Each centre processed the samples for FCI according to their usual strategies in their clinical practice, without previous agreement on standardization of the protocols used. All centres used the stain-lyse-wash protocol but panels of monoclonal antibodies were different: combinations of 4, 6 and 8 fluorochrome–conjugated monoclonal antibodies were included in 28, 8 and 12 files respectively. Median number of events recorded per file was 157,200 (range 10,000-500,000). The combination CD34/CD45/CD117 was included in 38 files and 20 also associated HLA-DR. The fluorochrome attached to CD34 was PerCP-Cy5 in 26 samples. 8G12 was the clone used in 40 samples. The degree of agreement among the 6 observers for quantification of CD34+ myeloid cells was evaluated using the intraclass correlation coefficient (ICC). The generalized kappa statistic for multiple rates (κ) calculated the concordance among observers after categorization of quantitative variables. Both the ICC and the generalized κ statistic were interpreted as follows: 0-0.2 poor; 0.3-0.4: fair; 0.5-0.6 moderate; 0.7-0.8 strong; >0.8 almost perfect agreement. Results Finally, 47 samples could be evaluated by the FCI observers. The ICC showed a strong agreement among observers (0.720), and also a good concordance on the quantification of CD34+ cells at the critical level of 2% (k=0.587). Regarding the comparison between FCI and morphology, only one participant counted >5% CD34+ cells in a sample. However, the absolute quantification of BM blasts <5% by FCI showed poor agreement with morphology (ICC ranged from 0.106 to 0.458). Indeed, none of the FCI observers could reproduce the new morphological categories using the threshold of 2% BM blasts (k= 0.320). Conclusions In our study, FCI seems a reproducible tool to quantifying CD34+ cells in MDS patients with <5% BM blasts, despite of the great heterogeneity of the protocols used. A FCI threshold of 2% CD34+ cells was also reproducible among observers. However, the lack of a precise correlation between the morphological blast cell count and the number of CD34+cells by FCI, illustrates the importance of considering each value independently. Probably, homogenization of the FCI protocols will contribute to improve the correlation between the 2 techniques. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Hesk Ratio: A Complementary Tool for the Diagnosis of Myelodysplastic Syndromes and a Novel Method for Flow Cytometry Analysis

Blood ◽

10.1182/blood.v120.21.4946.4946 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4946-4946

Author(s):

Evgenia Verigou ◽

Georgia Kolliopoulou ◽

Nikoleta Smirni ◽

Elisavet Hala ◽

Polixeni Lampropoulou ◽

...

Keyword(s):

Mathematical Model ◽

Bone Marrow ◽

Flow Cytometry ◽

High Risk ◽

Myelodysplastic Syndromes ◽

Myeloid Cells ◽

Antigen Expression ◽

Low Risk ◽

Empirical Parameter ◽

Analytical Approaches

Abstract Abstract 4946 Establishing the diagnosis of Myelodysplastic Syndromes (MDS) is a challenging task for hematologists due to the heterogeneity of this clinical entity. Several attempts have been made to include findings from advanced technologies to the diagnostic criteria of MDS, but still in the majority of cases, morphology of peripheral blood and bone marrow remains the cornerstone for the diagnosis. Flow cytometry(FC) can identify abnormal antigen expression on myeloid cells. FC has been proposed as a complementary method in the diagnosis of low and intermediate risk MDS, particularly for patients not exhibiting characteristic karyotype abnormalities. On the other hand, recent literature suggests that these findings are not MDS-related, questioning the specificity of immunophenotyping for the diagnosis of MDS. The aim of the present study is to maximize the utility of FC data and simplify their interpretation for the diagnosis of MDS, by developing new analytical approaches of digital data, other than the conventional sequential biparametric analysis. The applied methodology was based on a mathematical model of scale analysis. Bone marrow(BM) samples from 50 subjects were analysed for the expression of CD45PC7, CD11bPC5, CD16FITC and CD13PE (antigens by Beckman Coulter, FC500 flow cytometer Beckman Coulter). 36 patients were diagnosed with MDS (23 low risk, 13 high risk) and 14 patients had other than an MDS diagnosis (ITP, chronic idiopathic neutropenia, systemic lupus erythematosus, LGL leukemia, age-related cytopenias, aplastic anemia, myelofibrosis etc). Additionally, 3 BM samples of patients with post-MDS acute myeloid leukemia(AML) were analysed. The data used for the development of the mathematical model were the following: two populations (neutro1, neutron2) were gated according to their CD45 and CD13/CD16 antigen expression (Figure 1i-1v).Seven subpopulations of Neutrophils were defined on CD11b/CD16 density plot N=g+h+i and O=k+j (Figure 1vi). In an attempt to identify correlations between data that cannot be routinely revealed by sequential biparametric analysis, we have developed the HeSK* ratio, which is given by: where x is the median of CD11b in gate O, y is the median of CD16 in gate O, z is the median of CD45 in gate neutro, pO is the percentage of gate O in the total CD11b/CD16 diagram gated in neutro, pN is the percentage of gate N in the total CD11b/CD16 gated in neutro and 1000 is an empirical parameter. The HeSK ratio combines fluorescence levels of CD16, CD11b and CD45 with the percentage of two distinct neutrophil populations (N and O), which differ in their maturation and differentiation stage. The ratio can quantify the abnormal differentiation profile of mature myeloid cells and thus distinguish MDS from non-MDS samples with statistical significance P<0. 0001 (Kruskal Wallis test) as indicated in graph 1. Descriptive statistics are shown in table 1. · HeSK ratio is based upon a novel FC analysis method that could change the conventional biparametric routine FC analysis and quantify patterns that are not evaluated properly. Mathematical modeling of antigen expression patterns optimizes the interpretation of single immunophenotype findings. · The present study proposes HeSK as a complementary diagnostic tool for MDS and a strong indicator for the classification of the patients according to their prognosis as well. *the name HeSK comes from the initials of the 4 main authors (H=Hala, e=Evgenia, S=Smirni, K=Kolliopoulou). Table 1 non MDS low risk MDS high risk MDS Number of values 14 23 13 Minimum 50,76 4,789 0,2850 25% Percentile 304,8 26,11 17,05 Median 2133 92,52 47,64 75% Percentile 10650 228,9 144,3 Maximum 55040 3043 671,7 Mean 10320 316,1 122,7 Std. Deviation 17860 647,9 185,1 Std. Error 4773 135,1 51,33 Figure 1 Figure 1. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Analysis of the Immunophenotypic Features of Blast Cells, Lymphocytes and Granulocytes by Multiparametric Flow Cytometry in Bone Marrow (BM) Cells of Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v114.22.4843.4843 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4843-4843

Author(s):

Xin Du ◽

Jing Huang ◽

Maohua Zhou ◽

SuXia Geng ◽

Jianyu Weng ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Myelodysplastic Syndromes ◽

Flow Cytometric Analysis ◽

Control Group ◽

Consistent Finding ◽

Multiparametric Flow Cytometry ◽

Hla Dr ◽

Number Of Patients ◽

Blast Cells

Abstract Abstract 4843 Background The myelodysplastic syndromes (MDS) are a group of clonal heterogeneous bone marrow disorders characterized by peripheral cytopenias, ineffective hematopoiesis, and unilineage or multilineage dysplasia. Multiparametric flow cytometry is increasingly being used as an adjunct to the establishing of MDS. While many antigens have been described to be aberrantly expressed in MDS the findings are generally heterogeneous and there is no consistent finding that would be present in all cases with MDS. Aim To investigate the immunophenotypic features of MDS and non-MDS patients and the characteristic of subtypes of MDS. Methods BM samples were collected from 22 MDS patients including 3 RA (2 male, 1 female, median age 57), 3 RAS (2 male, 1 female, median age 72), 12 RAEB (6 male, 6 female, median age 67.5), 4 MDS-AML (2 male, 2 female, median age 69.5) and 20 non-MDS (11 male, 9 female, median age 32.5, 7 AA, 5 PNH, 3 IDA, 1 ALL, 2 CML, 2 MM). The multiparametric flow cytometric analysis was performed using an extensive panel of monoclonal antibodies and using the conventional and secondary gating strategies to analysis the immunophenotypic features of BM cells. Results This study showed that the proportion of blast cells increased significantly than non-MDS group (P=0.002). As the disease progressing, the percentage of blast cells became higher and significantly difference compared to the non-MDS group (P=0.226, P=0.464, P=0.001 and P=0.000, respectively). The expressions of CD34+ and CD7+ on blast cells were significantly difference between MDS and non-MDS groups (P=0.005, and P=0.002, respectively). Compared with the subtypes of MDS and non-MDS group, the expressions of CD34+ and CD7+ on blast cells became high gradually (P=0.534, P=0.487, P=0.009, P=0.004 and P=0.294, P=0.166, P=0.002, P=0.001) and the high percentage of blast cells and high expression levels of CD34+ and CD7+ might indicate poor prognosis. The expression of CD7+ on lymphocytes was similar with CD34+ and CD7+ on blast cells, but the expressions of CD19+ and CD56+ on lymphocytes were no significantly difference (P=0.076, P=0.252, respectively). The expressions of antigens on granulocytes showed that the expressions of CD15+CD11b+, CD10+ and HLA-DR were significantly difference between MDS and non-MDS groups(P=0.000, P=0.009 and P=0.007, respectively), meanwhile, as the disease progressing, the expression rates of CD15+CD11b+, CD10+ and HLA-DR in subtypes of MDS increased gradually and the survival time of these patients who had over-expression of these antigens was shorter than control group(P=0.002). However, the expressions of CD33+, CD13+, CD56+ and CD15+CD11b- were significantly difference between subtypes of MDS and non-MDS group (P=0.059, P=0.588, P=0.063 and P=0.207, respectively). Conclusions Our results showed that using multiparametric flow cytometry to analyze the immunophenotypic features of BM cells could provide clinically useful information for the diagnosis, classification and prognosis of MDS patients. Particularly, the percentage of blast cells, the expression of CD34+ and CD7+ on blast cells, the expression of CD7+ on lymphocytes and the expression of CD15+CD11b+, CD10+ and HLA-DR on granulocytes may provide the much more useful information. However, further studies including larger number of patients with a longer follow-up are necessary to confirm these results. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Decreased Expression of CD62L and CD54 Correlates with Poor Cytogenetic Risk Group In Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v116.21.2915.2915 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2915-2915

Author(s):

Canan Alhan ◽

Theresia M. Westers ◽

Claudia Cali ◽

Floortje L. Kessler ◽

Monique Terwijn ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Healthy Volunteers ◽

Board Of Directors ◽

Myelodysplastic Syndromes ◽

Research Funding ◽

Risk Category ◽

Advisory Committees ◽

Cd34 Cells

Abstract Abstract 2915 Interactions in the bone marrow (BM) between haematopoietic progenitor cells (HPC) and the BM micro environment are important for the regulation of cell adhesion, proliferation, differentiation and survival. Expression of both CD62L (L-selectin) and CD54 (ICAM-1) on HPC demonstrated to play a role in signal transduction routes for proliferation and growth regulation. Especially CD54 is involved in uncontrolled proliferation and block of apoptosis. Previously, it was described that decreased expression of CD62L in acute myeloid leukemia (AML) was associated with a poor cytogenetic risk profile and an adverse clinical outcome (Graf M et al, Eur J Haematol 2003) Myelodysplastic syndromes are a group of clonal HPC disorders characterized by ineffective hematopoiesis and a propensity to evolve into AML. The International Prognostic Scoring System (IPSS) provides information on both survival and risk of development of an AML. The purpose of our study was to evaluate CD62L and CD54 expression on CD34+ cells in MDS patients by flow cytometry and to assess the value of a CD62L/CD54 ratio for prognostication. Bone marrow samples of 30 newly diagnosed MDS patients (3 RA(RS)/18 RCMD(RS), the <5% blasts group; 5 RAEB-1, 4 RAEB-2, the >5% blasts group), 16 AML patients with prior MDS and 26 healthy volunteers were analyzed for CD62L and CD54 expression on CD34+ cells by using flow cytometry. An adhesion index was calculated as a ratio of the percentage and MFI of CD62L and CD54 positive cells (as was reported by Buccisano et al, Eur J Haematol 2007). The CD62L/CD54 ratio was significantly decreased in MDS with <5% blasts (median 79.09 p<0.0001) as compared to healthy volunteers (median 480.4) and even more decreased in high risk MDS (median 14.67 p<0.0001 and p=0.001 as compared to healthy volunteers and MDS with <5% blasts, respectively) and AML with prior MDS (median 12.54, p<0.0001 and p=0.009 as compared to healthy volunteers and MDS with <5% blasts, respectively). The MDS patients were assigned to the good, intermediate or poor IPSS cytogenetic risk category. Cytogenetics was available for 22 MDS patients. The CD62L/CD54 ratio was significantly lower in the cytogenetic poor risk category compared with the good risk category (median 5.4 and median 70.79 respectively, p=0.018). Moreover, a low CD62L/CD54 ratio correlated significantly with poor cytogenetics, p=0.006. In the group of MDS patients with <5% blasts, 4 developed a refractory anemia with excess of blasts or AML within a follow up period of 12 months. There was a trend for a lower CD62L/CD54 ratio for MDS patients who developed an AML compared with patients who did not. In conclusion, the CD62L/CD54 ratio is significantly decreased in MDS compared with healthy volunteers and even more decreased in AML with prior MDS. Both CD62L and CD54 are involved in regulation of proliferation and apoptosis of the HPC. A decreased adhesion ratio in low risk MDS patients might reflect HPC damage at an early stage of the disease with an increased proliferative capacity and a decreased apoptotic profile. Interestingly, a low CD62L/CD54 ratio showed a significant inverse correlation with the IPSS cytogenetic risk category. Due to an absence of metaphases in a proportion of MDS patients, cytogenetics is not always available. The CD62L/CD54 ratio might serve as a surrogate marker for poor prognosis cytogenetics in case no karyotype is available. Low risk MDS patients who developed an AML within 12 months tended to have a lower CD62L/CD54 ratio. Although these results are promising, sample size and follow up period needs to be extended. The CD62L/CD54 ratio might add to prognostication of MDS patients and might identify MDS patients with <5% blasts who are at risk for development of an AML. Disclosures: Ossenkoppele: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Van de Loosdrecht:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Dynamics of Bcl-2, Bax and ACE Expression In CD34+ Cells of Peripheral Blood and Bone Marrow In Patients with Acute Leukemia During Induction Therapy

Blood ◽

10.1182/blood.v116.21.4840.4840 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4840-4840

Author(s):

Elena E. Khodunova ◽

Elena N. Parovichnikova ◽

Irina V. Galtzeva ◽

Sergey M. Kulikov ◽

Valentin G. Isaev ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Drug Resistance ◽

Acute Leukemia ◽

Induction Therapy ◽

Peripheral Blood ◽

Acute Leukemias ◽

Healthy Donors ◽

Cd34 Cells ◽

Blast Cells

Abstract Abstract 4840 The causes of drug resistance in acute leukemias (AL) have been studied very intensively and the key research was done on Bcl-2 family proteins. Last studies have showed that high level Bcl-2 expression in acute leukemia is really associated with drug resistance andpoor prognosis [Haematologica 2007, U. Testa]. It was demonstrated that lower Bax/Bcl-2 ratio (<0,3) was associated with FAB M0-M1 classes (p=.00001), poor-risk cytogenetics and poor prognosis [Blood 2003, G. Poeta]. But there were no studies on the dynamic evaluation of Bcl2 and Bax expression on CD34+ cells during chemotherapy. Renin-angiotensin system and angiotensin concertin enzyme (ACE) influence on leukogenesis is extensively investigated. It was reported that ACE expression on blast cells is high [Leuk Lymphoma 2006, S. Aksu]. Recent publications indicate that primitive hematopoietic precursors have different characteristics regarding ACE: CD34+ACE+cells transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts and contain a significantly higher incidence of SCID-repopulating cells than the unfractionated CD34+ population [Blood 2008, V. Jokubaitis]. But it's still unknown how CD34+ACE+ cells in AL behave on and after chemotherapy. We have studied the dynamics of Bcl-2 and Bax expression by flow cytometry in CD34+ cells of peripheral blood (PB) and bone marrow (BM) in pts with AL. PB and BM samples were collected before treatment, on days +8, +36, only PB - on day + 21. Bcl-2 and Bax were detected on CD34+ cells by flow cytometry using specific monoclonal antibodies: CD34 (8G12, BD), Bcl-2 (100, BD), Bax (2D2, Santa Cruz). ACE (9B9, BD) expression was also evaluated. We calculated 10 000 cells in each sample. 10 pts were included in the study: 4 AML, 6 ALL. The control group comprised 4 healthy donors. At time of diagnosis CD34+ cells number in BM was 38,7%± 9,75, in PB - 38,3%± 8,14 in AL pts, not differing much in AML and ALL, and indicating blast cells population. CD34+ cells numbers in BM and PB of healthy donors were 1,35% and 0,23%, respectively. After induction therapy and WBC recovery (days +36-38) CD34+ cells number in AL pts decreased dramatically in BM to 3,83%±1,51 (p=0,001) and in PB to 0,98%± 0,29 (p=0,0001), indicating the efficacy of chemotherapy. The dynamics of Bcl-2, Bax and ACE expression on CD34+ cells of BM and PB in AL pts are presented in fig.1-6 As seen in the fig.1,2 CD34/Bcl-2 expression in BM is significantly higher (p=0,04) and in PB is similar in AL pts at the diagnosis comparing with donors. It's also worth to note that BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 demonstrating much higher level of antiapoptotic marker in PB cells. On the contrast CD34+ AL cells in BM and PB had similar characteristics regarding CD34/Bcl-2 expression. This expression level decreased substantially in BM at day +36 comparing with day 0 (p=0,04), but it never reached the donors level remaining extremely high and supposing the persistence of antiapoptotic activity in CD34+ cells in AL pts. It did not change at all during chemotherapy in PB cells, being identical to donors characteristics. The fig.2,3 demonstrate that, CD34/Bax expression in BM is almost 3-times higher (p=0,14) and in PB is twice lower (p=0,02) in AL pts in comparison with donors. It's interesting that CD34/Bax expression in leukemic BM and PB cells looks very similar, when in donors we had very low expression in BM and high - in PB. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar in Bax expression. Chemotherapy caused the significant augmentation of CD34/Bax expression in PB on day +8 (p=0,01) and near significant on day +21 (p= 0,09) showing the increased level of “dieing” cells in PB after cytostatic influence. The fig. 5,6 show that CD34/ACE coexpression in BM cells of AL pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AL pts was much lower (p=0,02) than in donors and substantially increased at day +36 almost reaching the donor level. We may conclude that Bcl-2, Bax, ACE expression on CD34+ cells in AL pts and donors significantly differs, the dynamics of expression in AL while chemotherapy shows critical changes in CD34/Bcl-2 expression in BM, CD34/Bax and CD34/ACE in PB. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

A Unified Prognostic Model for Myelodysplastic Syndrome and Acute Myeloid Leukaemia Based on Flow Cytometric Blast Count

Blood ◽

10.1182/blood.v118.21.1736.1736 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1736-1736

Author(s):

Alexandra Smith ◽

Simon Crouch ◽

Dan Painter ◽

Eve Roman ◽

Matt Cullen ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Cell Count ◽

Prognostic Model ◽

Blast Cell ◽

Bone Marrow Blast ◽

Flow Cytometric ◽

Blast Cells ◽

Blast Count ◽

Acute Myeloid

Abstract Abstract 1736 The distinction between Acute Myeloid Leukaemia (AML) and Myelodysplastic Syndrome (MDS), together with its prognostic assessment, depends on the proportion of bone marrow blast cells. The International Prognostic Scoring System (IPSS) uses this value to predict survival in conjunction with cytogenetic and blood count parameters. Current practice in most centres is to count blast cells manually on stained smears and to assign patients to one of 4 bands (<5%, 6–10%,11-19% and >20%). This approach has two major disadvantages. Firstly, the evaluation of cellular morphology is subjective and the standard error of a manual count is sufficiently large that many patients cannot be assigned with reasonable accuracy to a category - which can critically impact on their care. Secondly, assigning patients into broad groups, instead of retaining blast cell count as a continuous variable, may be degrading important prognostic information. Modern flow cytometric techniques allow blast cells to be counted with high levels of accuracy. In this study we used a standard five colour assay including CD34, CD117, CD15, CD3, CD19 and physical characteristics to count bone marrow blast cells. Bone marrows from 271 patients with AML/MDS treated with curative intent (median age, 59 yrs; M: F,1.22) and 684 patients treated with supportive therapy (median age, 77 yrs; M: F,1: 1.8) were evaluated. Patients with acute promyelocytic leukaemia (APML), AML with t(8;21) or inv 16 were excluded. All patients were from a population-based cohort (www.hmrn.org), and all diagnostic studies were performed using standard protocols in a single laboratory. A prognostic model was constructed using age, flow cytometric blast cell count and gender. In non-intensively treated patients survival declined rapidly with blast counts up to 10% and then decreases much more slowly. Age had a minimal effect on survival under the age of 70, but prognosis declined rapidly in older patients. Male gender was a significant adverse risk factor (hazard ratio = 1.47). In contrast, patients who were treated intensively showed only a modest relationship between blast count and prognosis but a much more marked effect of age. Survival declined progressively from 20 years with the trend accelerating in those over 60 years. There was also a smaller effect of gender in those receiving intensive treatment (hazard ratio = 1.09). In MDS patients, a complex relationship between blast cell count, standard cytogenetic risk factors and the degree of cytopenia was observed. Using age and flow cytometric blast count as continuous variables, modelled using restricted cubic splines, together with gender enabled more accurate outcome prediction in patients with AML and MDS across the full range of blast counts. This is possible because of the much higher levels of accuracy of flow cytometry compared to manual counting methods. The main practical advantage of a unified prognostic model is that it allows the relative benefits of intensive and non-intensive treatment to be readily compared for an individual patient. The predictive power of this core model can be improved further by the inclusion of additional clinical and molecular data. MDS and most forms of AML are part of a continuous spectrum of disease. A more unified approach to classification avoiding arbitrary subdivision may improve clinical decision making in this complex group of patients. Figure: The interrelationship between survival, age and blast cell count in patients with myelodysplastic syndrome. Figure:. The interrelationship between survival, age and blast cell count in patients with myelodysplastic syndrome. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

Post-Transplant Myelodysplastic Syndromes In Pediatric Liver Transplantation Recipients: a Report of Two Cases

Blood ◽

10.1182/blood.v116.21.4978.4978 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4978-4978

Author(s):

Estela Martin ◽

Silvia Grande ◽

M. Carmen Gomez del Castillo ◽

Rosa Ayala ◽

Ana Garcia Marcilla ◽

...

Keyword(s):

Bone Marrow ◽

Liver Transplantation ◽

Blood Cell ◽

Cell Count ◽

Organ Transplantation ◽

White Blood Cell Count ◽

Myelodysplastic Syndromes ◽

Bone Marrow Aspiration ◽

Blood Cell Count ◽

Blast Cells

Abstract Abstract 4978 Introduction: Organ transplantation recipients are generally considered to be at higher risk to develop a malignancy mainly due to prolonged inmunosuppression. The most common haematologic malignancies observed in the post-transplantation setting are B-cell lymphoproliferative disorders. Myeloid neoplasms are rare and most of them have been reported in patients undergoing heart, lung or kidney transplant. Only fifteen acute myeloid leukaemia (AML) and two myelodysplasic syndromes (MDS) cases have been described so far. We report the diagnosis of two MDS in two of the 160 paediatric recipients who underwent liver transplantation (LT) in our center. Patient 1: A 6-year-old child received a liver graft for idiopatic fulminant hepatic failure in December 2006. Inmunosuppressive medication was consisted of Basiliximab induction plus Tacrolimus and prednisone. On the eighth postoperative day a histologically proven rejection grade I occurred, successfully treated with methylprednisolone bolus therapy. Subsequently the patient did well until Abril 2010, when a peripheral blood smear showed 18% blast cells, 31% neutrophils, 46%lymphocytes. The white blood cell count was 4.2 × 1000/μL, the haemoglobin 10.6 g/dL and the platelets 34 × 1000/μL. Bone marrow aspiration showed dysplastic changes in the myelopoietic cells and 12% of blast cells. Myelodysplastic syndrome type refractory anemia with exess blasts (RAEB-2) was diagnosed. The blast cells inmunophenotype was CD34+, CD117+, CD13++, and CD33+. Kariotype analysis revealed a normal 46XX kariotype. Patient 2: In May 2007 a 15-year-old female patient underwent LT for idiopathic acute liver failure. Inmunosuppression consisted of tacrolimus and steroids for the first 3 months. No induction therapy was added. The patient remained in good health with good liver function for 31 months. In a routine follow-up in December 2009 pancytopenia was detected. The white blood cell count was 3.1 × 1000/μL with 31% neutrophils, 66% lymphocytes and 2% monocytes. The haemoglobin was 7.7 g/dL, and platelets were 29 × 1000/μL. Subsequent bone marrow aspiration showed multilineage dysplasia and 15% myeloid blast cells, classified as MDS type RAEB-2. No HLA related donor was found. Two months later the bone marrow showed leukemic infiltration with 38% myeloid blast cells. The inmunophenotype was CD34+, CD117+, CD13+, CD33+, CD133+, and MPO+. Cytogenetic analysis revealed a normal 46XX kariotype. AML with myelodysplasia-related changes was diagnosed. No molecular changes typical of therapy-related MDS were found in any of the cases. Donor and recipient genotypes were identified by real-time PCR to quantify donor chimerism (DC) after LT. In the first case, there was no difference between the donor and recipient genotype and chimerism study could not be done. In the second case DC after LT was not found neither at the moment of the MDS diagnose nor in the next 3 months. Conclusion: MDS/AML has rarely been reported after liver transplantation, and MDS isn't frequently found in the paediatric population. In contrast to previous reports rapid progression to AML was observed in our patients. The present cases support the need for further investigation of the mechanism leading to post-transplant myelodysplastic syndromes and the acute leukaemia in the setting of solid organ transplantation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text