RESUMO
The assessment of bone marrow iron stores is typically performed on an aspirate smear slide that has been manually stained by a technologist using a commercially available kit. This approach can contribute to inconsistent results and limit the broad use of iron staining in bone marrow specimens, particularly when laboratories have low staffing and/or high specimen volumes. Here, we describe the adaptation and validation of the Ventana Benchmark automated stainer and iron stain kit for routine clinical use of staining iron in bone marrow aspirate smear slides. We assessed accuracy and precision of the Ventana automated iron staining protocol compared to the Perls Prussian blue manual iron staining index method. Hematopathologists assigned Gale scores and enumerated the percentages of erythroid sideroblasts on paired patient bone marrow aspirate smear slides stained by the automated method and the manual iron staining method. We found a similar level of performance of the Ventana automated iron stain relative to the index manual method (as assessed by Pearson correlation and Bland-Altman analyses). In addition, there was low imprecision between replicates performed via the automated iron stain protocol. We also report superior qualitative findings of the automated method in ease of localization of iron storage, visualization of sideroblasts, and counterstain consistency. Automated iron staining of bone marrow aspirate smear slides performed similarly to the manual method and may allow for accurate routine evaluation of bone marrow iron stores as part of bone marrow analysis.
Assuntos
Medula Óssea , Ferro , Coloração e Rotulagem , Humanos , Ferro/análise , Ferro/metabolismo , Coloração e Rotulagem/métodos , Medula Óssea/patologia , Medula Óssea/metabolismo , Exame de Medula Óssea/métodos , Automação Laboratorial , Reprodutibilidade dos Testes , Células da Medula Óssea/metabolismoRESUMO
BACKGROUND: Involvement of the central nervous system (CNS) by acute leukemias (ALs) has important implications for risk stratification and disease outcome. The clinical laboratory plays an essential role in assessment of cerebrospinal fluid (CSF) specimens from patients with ALs at initial diagnosis, at the end of treatment, and when CNS involvement is clinically suspected. The two challenges for the laboratory are 1) to accurately provide a cell count of the CSF and 2) to successfully distinguish blasts from other cell types. These tasks are classically performed using manual techniques, which suffer from suboptimal turnaround time, imprecision, and inconsistent inter-operator performance. Technological innovations in flow cytometry and hematology analyzer technology have provided useful complements and/or alternatives to conventional manual techniques. AIMS: We performed a PRISMA-compliant systematic review to address the medical literature regarding the development and current state of the art of CSF blast identification using flow cytometry and laboratory hematology technologies. MATERIALS AND METHODS: We searched the peer reviewed medical literature using MEDLINE (PubMed interface), Web of Science, and Embase using the keywords "CSF or cerebrospinal" AND "blasts(s)". RESULTS: 108 articles were suitable for inclusion in our systematic review. These articles covered 1) clinical rationale for CSF blast identification; 2) morphology-based CSF blast identification; 3) the role of flow cytometry; 4) use of hematology analyzers for CSF blast identification; and 5) quality issues. 9 /L, which is much lower than the original machine count and platelet transfusion was warranted. DISCUSSION: 1) Clinical laboratory testing plays a central role in risk stratification and clinical management of patients with acute leukemias, most clearly in pediatric ALs; 2) studies focused on other patient populations, including adults and patients with AML are less prevalent in the literature; 3) improvements in instrumentation may provide better performance for the classification of CSF specimens. CONCLUSION: Current challenges include: 1) more precisely characterizing the natural history of AL involvement of the CNS, 2) improvements in automated cell count technology of low cellularity specimens, 3) defining the role of flow MRD testing of CSF specimens and 4) improved recognition of specimen quality by clinicians and laboratory personnel.
Assuntos
Hematologia , Leucemia , Adulto , Líquido Cefalorraquidiano , Criança , Citometria de Fluxo/métodos , Hematologia/métodos , Humanos , Contagem de Leucócitos , TecnologiaAssuntos
Linfócitos B/patologia , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Haploinsuficiência , Síndromes Mielodisplásicas/patologia , Células-Tronco/patologia , Animais , Linfócitos B/metabolismo , Humanos , Camundongos , Camundongos Knockout , Síndromes Mielodisplásicas/genética , Células-Tronco/metabolismoRESUMO
Next generation sequencing (NGS) methods have allowed for unprecedented genomic characterization of acute myeloid leukemia (AML) over the last several years. Further advances in NGS-based methods including error correction using unique molecular identifiers (UMIs) have more recently enabled the use of NGS-based measurable residual disease (MRD) detection. This review focuses on the use of NGS-based MRD detection in AML, including basic methodologies and clinical applications.
RESUMO
Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.
Assuntos
Apoptose/genética , Proteínas de Choque Térmico HSP70/deficiência , Proteínas de Choque Térmico HSP70/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Biomarcadores , Resistência a Medicamentos/genética , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Mitocondriais/antagonistas & inibidores , Síndromes Mielodisplásicas/genética , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tiazóis/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Somatic mutations in spliceosome genes are detectable in â¼50% of patients with myelodysplastic syndromes (MDS). We hypothesize that cells harbouring spliceosome gene mutations have increased sensitivity to pharmacological perturbation of the spliceosome. We focus on mutant U2AF1 and utilize sudemycin compounds that modulate pre-mRNA splicing. We find that haematopoietic cells expressing mutant U2AF1(S34F), including primary patient cells, have an increased sensitivity to in vitro sudemycin treatment relative to controls. In vivo sudemycin treatment of U2AF1(S34F) transgenic mice alters splicing and reverts haematopoietic progenitor cell expansion induced by mutant U2AF1 expression. The splicing effects of sudemycin and U2AF1(S34F) can be cumulative in cells exposed to both perturbations-drug and mutation-compared with cells exposed to either alone. These cumulative effects may result in downstream phenotypic consequences in sudemycin-treated mutant cells. Taken together, these data suggest a potential for treating haematological cancers harbouring U2AF1 mutations with pre-mRNA splicing modulators like sudemycins.
Assuntos
Cicloexilaminas/farmacologia , Mutação , Splicing de RNA/efeitos dos fármacos , Compostos de Espiro/farmacologia , Spliceossomos/efeitos dos fármacos , Fator de Processamento U2AF/genética , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Cultura Primária de Células , Spliceossomos/genética , Fator de Processamento U2AF/metabolismoRESUMO
Heterozygous somatic mutations in the spliceosome gene U2AF1 occur in â¼ 11% of patients with myelodysplastic syndromes (MDS), the most common adult myeloid malignancy. It is unclear how these mutations contribute to disease. We examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model. Mice expressing mutant U2AF1(S34F) display altered hematopoiesis and changes in pre-mRNA splicing in hematopoietic progenitor cells by whole transcriptome analysis (RNA-seq). Integration with human RNA-seq datasets determined that common mutant U2AF1-induced splicing alterations are enriched in RNA processing genes, ribosomal genes, and recurrently mutated MDS and acute myeloid leukemia-associated genes. These findings support the hypothesis that mutant U2AF1 alters downstream gene isoform expression, thereby contributing to abnormal hematopoiesis in patients with MDS.