Multicenter Comparison of CD34+ Myeloid Cell Count by Flow Cytometry in Low-Risk Myelodysplastic Syndrome. Is It Feasible?

2017 ◽  
Vol 55 ◽  
pp. S98-S99
Author(s):  
P. Font ◽  
D. Subira ◽  
S. Matarraz ◽  
C. Benavente ◽  
T. Cedena ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndrome ◽  
Cell Count ◽  
Myeloid Cell ◽  
Low Risk

scholarly journals Multicenter comparison of CD34+ myeloid cell count by flow cytometry in low-risk myelodysplastic syndrome. Is it feasible?

2017 ◽  
Vol 94 (3) ◽  
pp. 527-535 ◽  
Author(s):  
Patricia Font ◽  
Dolores Subirá ◽  
Sergio Matarraz ◽  
Celina Benavente ◽  
María Teresa Cedena ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Myelodysplastic Syndrome ◽  
Cell Count ◽  
Myeloid Cell ◽  
Low Risk

Flow Cytometric Detection of Human Telomerase Reverse Transcriptase Expression in CD34 Positive Precursor Fraction in Myelodysplastic Syndrome Bone Marrow.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4300-4300
Author(s):  
Hiroshi Handa ◽  
Takafumi Matsushima ◽  
Norifumi Tsukamoto ◽  
Masamitsu Karasawa ◽  
Hiroyuki Irisawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Disease Progression ◽  
Risk Group ◽  
Telomerase Activity ◽  
Low Risk ◽  
Htert Expression ◽  
Trap Assay

Abstract Telomerase activity has been found in most common cancers indicating that telomerase detection may be a useful marker in cancer diagnosis. For detection of telomerase activity and the expression of associated genes in cells, TRAP assay and RT-PCR are customarily used. Immunohistochemical detection of hTERT is useful to detect telomerase-positive cells in a background of non- cancerous cells. We developed a method for the detection of intra-nuclear hTERT protein, in a sub-population of hematopoietic cells, using concurrent staining cell surface antigen and multi color flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas neutrophils of normal subjects showed 0% positivity, it is consistent with telomerase activity assessed by TRAP assay (r=0.71, p<0.0001) and previous observations. Then we applied this method to analyze hTERT expression in myelodysplastic syndrome (MDS). Forty MDS patients samples were obtained, 36 patients were diagnosed as low risk MDS (RA), 14 patients were diagnosed as high risk MDS (RAEB or RAEB-t) according to FAB classification. All samples were acquired after informed consent was obtained from the patients. Expression of hTERT protein was higher in CD34-positive blast-gated cells than CD34-negative blast-gated cells. The percentage of the CD34+ cells expressing hTERT ranged from 9.66% to 90.91% in low risk MDS patients, whereas from 50.46% to 97.68% in high risk MDS. The expression level was higher in the high risk group compared to that in the low risk group in MDS (p=0.0054, p=0.0084). This observation implied that telomerase up-regulation and hTERT expression were important for disease progression and could be a marker of more advanced disease. In subsets of MDS and AML bone marrow specimens obtained from these patients, we examined the hTERT expression in CD34+/CD38 high cells and CD34+/CD38 low cells containing stem cell fraction. Of interest, some of the patients showed higher expression of hTERT in CD34+/CD38 low cells than in CD34+/CD38 high cells. This observation is inconsistent with previous reports describing normal bone marrow hematopoietic cell findings. We speculated that this phenomenon could be a marker of MDS abnormality and that telomerase up-regulation may be initiated in the more primitive precursor fraction containing hematopoietic stem cells during the disease progression. Telomerase studies may be useful for definition of the risks associated with disease severity. Multi-parameter nature of flow cytometry and its ability to identify cellular sub-populations will facilitate a fuller understanding of the mechanisms of activation of telomerase.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2769-2769
Author(s):  
Patricia Font ◽  
Dolores Subirá ◽  
Sergio Matarraz ◽  
Celina Benavente ◽  
Teresa Cedena ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Cell Count ◽  
Myelodysplastic Syndromes ◽  
Blast Cell ◽  
Low Risk ◽  
Cd34 Cells ◽  
Blast Cells ◽  
Good Concordance

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.

Reduced expression of FLIPSHORT in bone marrow of low risk myelodysplastic syndrome

2007 ◽  
Vol 31 (6) ◽  
pp. 853-857 ◽  
Author(s):  
Paula de Melo Campos ◽  
Fabíola Traina ◽  
Adriana da Silva Santos Duarte ◽  
Irene Lorand-Metze ◽  
Fernando F. Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Low Risk

scholarly journals Study of Peripheral Mononuclear Cells and CD34 Levels as a Predictive Marker for Initiating Apheresis in Autologous Stem Cell Transplant

2021 ◽  
Author(s):  
Kiran PK ◽  
Vinu Sarathy P ◽  
Srinivas BJ ◽  
Girish V Badarkhe ◽  
Rajesh Kumar KS ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Cell Count ◽  
Hodgkin Lymphoma ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Blood Stem Cell ◽  
Optimal Time ◽  
Cell Transplant ◽  
Stem Cell Collection

Background: Autologous HCT in multiple myeloma is done as upfront treatment in newly diagnosed transplant eligible patients after induction chemotherapy. In addition, it is standard for relapsed, aggressive non-Hodgkin lymphoma (NHL) and classical Hodgkin lymphoma (HL), and is curative in ~40% to 45% of patients. Over a decade, many efforts were made to find helpful parameters to predict an optimal time for initiating an efficient peripheral blood stem cell collection so that adequate stem cells are collected.  It has been well accepted that CD34+ cell count in peripheral blood before leukapheresis is the best parameter to predict CD34 cell yield. However, white blood cell count, mononuclear cell count, and other easily obtained parameters are still used to guide the clinical practice of peripheral blood stem cell mobilization and collection.  Materials and Methods: In the present study, we analyzed the correlation between peripheral blood MNC and Apheresis CD34 levels and also between peripheral blood CD34 by flow cytometry and apheresis CD34 levels. Results: We found that there was a statistically insignificant weak correlation between peripheral MNC and apheresis CD34. There was a statistically significant strong correlation between peripheral CD34 and apheresis CD34. Conclusion: The results show that peripheral blood MNC was analogous indicating that no reliable prediction can be done for CD34 cells collected in apheresis while peripheral CD34 by flow cytometry is the strongest predictor for initiating stem cell collection.

scholarly journals Mutational Characteristics and Changing Pattern from Idiopathic Cytopenia of Undetermined Significance to High-Risk Myelodysplastic Syndrome Stratified By IPSS-R

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3009-3009
Author(s):  
Eun-Ji Choi ◽  
Young-Uk Cho ◽  
Seongsoo Jang ◽  
Chan-jeoung Park ◽  
Han-Seung Park ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Myelodysplastic Syndrome ◽  
Rna Splicing ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
International Prognostic Scoring System ◽  
Intermediate Risk ◽  
Sequencing Data ◽  
Undetermined Significance

Background: Unexplained cytopenia comprises a spectrum of hematological diseases from idiopathic cytopenia of undetermined significance (ICUS) to myelodysplastic syndrome (MDS). Revised International Prognostic Scoring System (IPSS-R) is the standard tool to assess risk in MDS. Here, we investigated the occurrence, characteristics, and changing pattern of mutations in patients with ICUS and MDS stratified by IPSS-R score. Methods: A total of 211 patients were enrolled: 73 with ICUS and 138 with MDS. We analyzed the sequencing data of a targeted gene panel assay covering 141 genes using the MiSeqDx platform (Illumina). The lower limit of variant allele frequency (VAF) was set to 2.0% of mutant allele reads. Bone marrow components were assessed for the revised diagnosis according to the 2016 WHO classification. Lower-risk (LR) MDS was defined as those cases with very low- or low-risk MDS according to the IPSS-R. Higher-risk (HR) MDS was defined as those cases with high- or very high-risk MDS according to the IPSS-R. Results: Patients with ICUS were classified as very low-risk (39.7%), low-risk (54.8%), and intermediate-risk (5.5%) according to the IPSS-R. Patients with MDS were classified as LR (35.5%), intermediate-risk (30.4%), and HR (34.1%). In the ICUS, 28 (38.4%) patients carried at least one mutation in the recurrently mutated genes in MDS (MDS mutation). The most commonly mutated genes were DNMT3A (11.0%), followed by TET2 (9.6%), BCOR (4.1%), and U2AF1, SRSF2, IDH1 and ETV6 (2.7% for each). IPSS-R classification was not associated with mutational VAF and the number of mutations in ICUS. In the 49 LR MDS, 28 (57.1%) patients carried at least one MDS mutation. The most commonly mutated genes were SF3B1 (20.4%), followed by TET2 (12.2%), U2AF1 (10.2%), DNMT3A (10.2%), ASXL1 (10.2%), and BCOR (6.1%). Higher VAF and number of mutations were observed in LR MDS compared to ICUS patients. In the 42 intermediate-risk MDS, 27 (64.3%) patients carried at least one MDS mutation. The most commonly mutated genes were ASXL1 (23.8%), followed by TET2 (21.4%), RUNX1 (16.7%), U2AF1 (14.3%), DNMT3A (14.3%), SF3B1 (9.5%), and SRSF2, BCOR, STAG2 and CBL (7.1% for each). In the 47 HR MDS, 36 (76.6%) patients carried at least one MDS mutation. The most commonly mutated genes were TET2 (25.5%), followed by DNMT3A (14.9%), TP53 (14.9%), RUNX1 (12.8%), U2AF1 (10.6%), ASXL1 (10.6%), and SRSF2 and KRAS (6.4% for each). As the disease progressed, VAF and number of the MDS mutations gradually increased, and mutations involving RNA splicing, histone modification, transcription factor or p53 pathway had a trend for increasing frequency. Specifically, ASXL1, TP53, and RUNX1 mutations were the most striking features in patients with advanced stage of the disease. Cohesin mutations were not detected in ICUS, whereas these mutations were detected at a relatively high frequency in HR MDS. Our data were summarized in Table 1. Conclusions: We demonstrate that on disease progression, MDS mutations are increased in number as well as are expanded in size. Furthermore, a subset of mutations tends to be enriched for intermediate- to HR MDS. The results of this study can aid both diagnostic and prognostic stratification in patients with unexpected cytopenia. In particular, characterization of MDS mutations can be useful in refining bone marrow diagnosis in challenging situations such as distinguishing LR MDS from ICUS. Disclosures No relevant conflicts of interest to declare.

ATM polymorphism is associated with low risk myelodysplastic syndrome

DNA Repair ◽  
2013 ◽  
Vol 12 (2) ◽  
pp. 87-89 ◽  
Author(s):  
Howard Lopes Ribeiro ◽  
Roberta Taiane Germano De Oliveira ◽  
Allan Rodrigo Soares Maia ◽  
Juliana Cordeiro De Sousa ◽  
Fabiola Fernandes Heredia ◽  
...  
Keyword(s):  
Myelodysplastic Syndrome ◽  
Low Risk

scholarly journals GPX3 methylation is associated with hematologic improvement in low-risk myelodysplastic syndrome patients treated with Pai-Neng-Da

2020 ◽  
Vol 48 (9) ◽  
pp. 030006052095689
Author(s):  
Shujun Yang ◽  
Tong Gao ◽  
Zhonghua Zheng ◽  
Binbin Lai ◽  
Lixia Sheng ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Myelodysplastic Syndrome ◽  
Methylation Level ◽  
Hematological Parameters ◽  
Low Risk ◽  
Controlled Clinical Trial ◽  
Platelet Response ◽  
Female Patients ◽  
Platelet Counts ◽  
Male Patients

Objective The aim of this prospective randomized controlled clinical trial was to explore the relationship between GPX3 methylation and Pai-Neng-Da (PND) in the treatment of patients with low-risk myelodysplastic syndrome (MDS). Methods There were 82 low-risk MDS patients who were randomly divided into the following two groups: androl, thalidomide, and PND capsule (ATP group, n = 41); or androl and thalidomide (AT group, n = 41). Hemoglobin and neutrophil and platelet counts and changes in GPX3 methylation level were assessed. Results The plasma hemoglobin level increased in both groups after treatment. However, the platelet count increased only in the ATP group. Patients in the ATP group had a better platelet response than the AT group, and GPX3 methylation markedly decreased after treatment with ATP but not after treatment with AT. Moreover, male patients had a significantly lower GPX3 methylation level than female patients, while platelet counts from male patients increased dramatically after the ATP regimens compared with female patients. GPX3 methylation changes were negatively correlated with platelet changes in ATP group. Conclusion PND can improve hematological parameters and decrease the GPX3 methylation level. Decreasing GPX3 methylation is associated with the hematologic response that includes platelet in GPX3 methylation. China Clinical Trial Bureau (ChiCTR; http://www.chictr.org.cn/ ) registration number: ChiCTR-IOR-15006635.

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