Rapid and Simple Isolation and Concentration Procedure for Human Megakaryocytes

人巨核细胞快速、简单的分离和浓缩程序

• 批准号: 8952876
• 负责人: Raul Braylan
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8952876
• 关键词: Acute Megakaryocytic Leukemias; Affect; Age; Antibodies; Antigens; Aspirate substance; Biochemical; Biopsy; Blood; Blood Platelets; Bone Marrow; Buffers; Cell Survival; Cells; Classification; Clinical; Complex; DNA Binding; Diagnostic; Diagnostic tests; Disease; Dysmyelopoietic Syndromes; Evaluation; Face; Filtration; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescence-Activated Cell Sorting; Goals; Heterogeneity; Human; Idiopathic Thrombocytopenic Purpura; Immune; Immunophenotyping; In Situ Hybridization; Incidence; Institution; Jordan; Label; Laboratories; Large Megakaryocyte; Left; Lymphoid Cell; Manuals; Marrow; Medical center; Megakaryocytes; Methods; Microscopic; Military Personnel; Molecular; Molecular Probes; Monitor; Myeloproliferative disease; Pathology; Patients; Ploidies; Polyploid Cells; Population; Preparation; Procedures; Production; Recovery; Relative (related person); Saline; Sampling; Signal Transduction; Staining method; Stains; Surface Antigens; Suspension substance; Suspensions; Techniques; Technology; Testing; Time; United States National Institutes of Health; Work; abstracting; analytical method; base; density; disease diagnosis; human ITGB3 protein; magnetic beads; rapid detection; research study; volunteer

Background Megakaryocytes (MK) comprise a rare cell population in the bone marrow, making up an estimated 0.1-0.5% of the total nucleated cells. Numerical and morphologic abnormalities of MK are described in a variety of primary and secondary marrow disorders but the scarcity of these cells makes analysis difficult and enrichment techniques for subsequent cell analysis are complex and labor intensive. Currently, MK changes are assessed exclusively on microscopic preparations (marrow aspirates and biopsies), and are used as important criteria for disease diagnosis, classification and therapy monitoring despite the inherent subjectivity of microscopic evaluations. In contrast to other hemopoietic or lymphoid cells, adequate quantitative studies of freshly isolated MK have proven difficult because of the relative rarity of these cells. Even flow cytometry (FCM), which is ideal for rapid detection of infrequent cell populations, faces serious difficulties in the assessment of marrow MK and the analysis of these cells is not part of the routine diagnostic work up of marrow cells in clinical laboratories. MK can be isolated using separation techniques such as density gradients, magnetic beads, centrifugal elutriation or fluorescence activated cell sorting, but these are labor-intensive, time-consuming or costly methods. Mature marrow MK are large, polyploid cells and it has been shown that their size distribution overlaps minimally with that of all other marrow cells. This distinct size threshold implies that size alone is a discriminatory parameter for MK isolation. Thus, we developed a simple and inexpensive manual mesh filtration method for separation of MK that allows a rapid and easy size-based concentration and purification of these cells. Materials and Methods We examined 15 discarded anonymized BM aspirate samples suitable for our analysis. These samples were from patients with a variety of hematologic disorders originally submitted to our laboratory for evaluation. Sample age ranged from 1-4 days (mean 1.5 days). Samples were diluted with a buffered saline solution and the cell suspensions were filtered through meshes of appropriate size. This filtration procedure allowed the preferential retention of MKs and, on average, the procedure only lasted 15 minutes. The resulting cell suspensions were stained with FITC-anti CD61 or PE-anti CD41a antibodies Becton Dickinson (BD) to label MKs, concurrently with the cell permeable DNA-binding DRAQ5 (Cell Signaling Technology) to identify all nucleated cells. Final cell viability was assessed by C12Resazurin (Molecular probes). Cell analysis was performed by flow cytometry (FCM) using a CANTO II flow cytometer (BD). Absolute MK enumeration was performed using the Flow Cytometry Absolute Count Standard beads (Bangs). MK enrichment efficiency was expressed as the percentage of MKs of all nucleated cells determined by FCM. Results and Conclusions The median (range) volume of the BM samples used in this study was 0.54 (0.24-1.99) mL. MKs were identified by FCM on the basis of their large size, expression of platelet-associated antigens and DNA ploidy levels. The median (range) MK recovery was 31 (14-100) % of the original number of MKs and the yield was 9,882 (1,519-49,921) MKs per mL of BM aspirate. The median (range) fraction of MKs among all nucleated cells after filtration was 39 (14-68) %, representing a 904 (439-3029)-fold MK enrichment. The MK viability after filtration was near 100%. This rapid and inexpensive isolation and concentration method results in a MK recovery and purification that is comparable or better than other more elaborate techniques. Despite the inherent heterogeneity of the samples used, we obtained a reasonably good recovery of MKs per mL of marrow aspirate and more than 900-fold median MK concentration. The yield and level of purification of freshly isolated MKs obtained by thiiis simple procedure may be useful in studies of a variety of primary or secondary marrow disorders. In particular, it should facilitate the application of analytical methods such as flow cytometry or in situ hybridization, and even be useful for biochemical or molecular testing that requires adequate cell representation and purity. Current Studies Using flow cytometry, we are presently determining DNA content (ploidy) and expression of a variety of surface antigens in MKs isolated from fresh aspirates as described above in marrows from normal volunteers and patients with a variety of hematologic disorders. The goal of this study is to establish changes in MK ploidy and/or immunophenotype, if any, in diseases in which MK are expected to be abnormal based on morphologic grounds, such as myelodysplasias or myeloproliferative neoplasms. Unfortunately, the incidence of these disorders at the NIH is currently low. Furthermore, hematologic patients with marrow disorders in this institution undergo extensive research studies and diagnostic testing that require a significant amount of marrow sample, frequently leaving a relatively scanty amount of material for our studies. For this reason, we contacted professionals in the Department of Pathology of the Walter Reed National Military Medical Center who have access to marrow samples, in order to establish a collaborative effort to obtain adequate and representative material for our studies. Abstract: Raul C Braylan, Elaine Kay Jordan. Rapid and Simple Procedure For Isolation and Concentration Of Human Megakaryocytes From Marrow Aspirates. Blood 2013 122:5269

背景 巨核细胞（MK）是骨髓中一种罕见的细胞群，估计占总有核细胞的0.1-0.5%。MK的数量和形态异常在各种原发性和继发性骨髓疾病中有描述，但这些细胞的稀缺性使得分析困难，并且用于后续细胞分析的富集技术复杂且劳动密集。 目前，MK变化仅在显微镜制备物（骨髓抽吸物和活检）上进行评估，并且被用作疾病诊断、分类和治疗监测的重要标准，尽管显微镜评估具有固有的主观性。与其他造血或淋巴细胞相比，由于这些细胞相对稀少，对新鲜分离的MK进行充分的定量研究已被证明是困难的。即使是流式细胞术（FCM），这是理想的快速检测罕见的细胞群，面临着严重的困难，在评估骨髓MK和分析这些细胞不是一部分的常规诊断工作的骨髓细胞在临床实验室。 MK可以使用分离技术如密度梯度、磁珠、离心淘洗或荧光激活细胞分选来分离，但这些都是劳动密集型、耗时或昂贵的方法。成熟骨髓MK是大的多倍体细胞，并且已经显示它们的大小分布与所有其他骨髓细胞的大小分布重叠最小。这种独特的大小阈值意味着大小本身是MK分离的判别参数。因此，我们开发了一种简单且廉价的手动筛网过滤方法用于分离MK，其允许快速且容易的基于大小的这些细胞的浓缩和纯化。 材料和方法 我们检查了15个丢弃的匿名骨髓穿刺样本，适合我们的分析。 这些样本来自最初提交给我们实验室进行评价的各种血液学疾病患者。 样本存放时间为1-4天（平均1.5天）。 用缓冲盐水溶液稀释样品，并通过适当尺寸的筛网过滤细胞悬液。 该过滤程序允许优先保留MK，并且该程序平均仅持续15分钟。 用FITC-抗CD 61或PE-抗CD 41 a抗体Becton Dickinson（BD）对所得细胞悬浮液进行染色以标记MK，同时用细胞可渗透的DNA结合DRAQ 5（Cell Signaling Technology）鉴定所有有核细胞。 通过C12刃天青（分子探针）评估最终细胞活力。 使用CANTO II流式细胞仪（BD）通过流式细胞术（FCM）进行细胞分析。 使用流式细胞术绝对计数标准微珠（Bangs）进行MK绝对计数。 MK富集效率表示为通过FCM测定的所有有核细胞中MK的百分比。 结果和结论 本研究中使用的BM样本的中位（范围）体积为0.54（0.24-1.99）mL。 通过流式细胞术根据其大的尺寸、血小板相关抗原的表达和DNA倍体水平鉴定MK。中位（范围）MK回收率为原始MK数量的31（14-100）%，产量为9，882（1，519 - 49，921）MK/mL BM抽吸物。 过滤后所有有核细胞中MK的中位数（范围）分数为39（14-68）%，代表MK富集904（439-3029）倍。 过滤后MK活力接近100%。这种快速和廉价的分离和浓缩方法导致MK回收和纯化，其与其他更复杂的技术相当或更好。 尽管所用样本存在固有的异质性，但我们获得了相当好的每mL骨髓穿刺液MK回收率和超过900倍的MK浓度中位数。 通过这种简单的方法获得的新鲜分离的MK的产率和纯化水平可用于各种原发性或继发性骨髓疾病的研究。 特别是，它应该有利于应用分析方法，如流式细胞术或原位杂交，甚至是有用的生化或分子测试，需要足够的细胞代表性和纯度。 目前的研究 使用流式细胞术，我们目前正在测定从正常志愿者和患有各种血液疾病的患者的骨髓中的新鲜抽吸物分离的MK中的DNA含量（倍性）和各种表面抗原的表达，如上所述。 本研究的目的是确定MK倍性和/或免疫表型的变化，如果有的话，在疾病中，MK是基于形态学的理由，如骨髓增生异常或骨髓增生性肿瘤异常。 不幸的是，这些疾病在NIH的发病率目前很低。 此外，该机构中患有骨髓疾病的血液病患者进行广泛的研究和诊断测试，需要大量的骨髓样本，通常会为我们的研究留下相对较少的材料。出于这个原因，我们联系了沃尔特里德国家军事医学中心病理学部门的专业人员，他们可以获得骨髓样本，以便建立合作努力，为我们的研究获得足够的和有代表性的材料。 翻译后摘要：劳尔C Braylan，伊莱恩凯约旦。 从骨髓穿刺液中分离和浓缩人巨核细胞的快速简便方法。 第122章：你是谁？