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: 10913206
• 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/10913206
• 关键词: Abnormal megakaryocyte; Acute Megakaryocytic Leukemias; Acute Myelocytic Leukemia; Affect; Antigens; Biopsy; Blood Platelets; Bone Marrow; Bone Marrow Aspiration; Buffers; Cell Lineage; Cell Maturation; Cells; Classification; Clinical; Collecting Cell; Complex; Consumption; DNA analysis; Data; Diagnostic; Disease; Dysmyelopoietic Syndromes; Electron Microscopy; Elements; Evaluation; Excision; Exhibits; Face; Filtration; Flow Cytometry; Fluorescence-Activated Cell Sorting; Frequencies; Germany; Glutaral; Goals; Hematological Disease; Hematology; Hematopoietic; Heterogeneity; Human; ITGB3 gene; Idiopathic Thrombocytopenic Purpura; Immune; Label; Laboratories; Large Megakaryocyte; Lobe; Lymphoid Cell; Manuals; Marrow; Megakaryocytes; Methods; Microscopic; Monitor; Morphology; Myeloproliferative disease; Nuclear; Nylons; Patients; Ploidies; Polyploid Cells; Population; Preparation; Procedures; Process; Production; Proliferating; Proliferation Marker; Sample Size; Sampling; Stains; Suspensions; Techniques; Time; Visual; Wright-Giemsa Staining Method; aspirate; cost; cytopenia; density; diagnostic value; disease diagnosis; magnetic beads; rapid detection

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 For the MK enrichment, discarded bone marrow aspirates from patients with a variety of hematologic disorders were diluted with a buffered solution (8% BSA in HBSS) and the cell suspensions were first passed through a 100m filter prior to micro-filtration. For the micro-filtration, we initially used a 8 90mm Circular Nylon Mesh Filter (Repligen) but because of the discontinuation of this filter, we switched to a 2-stage filtration with a 10m and a 5m mesh from Pluriselect (Germany) made of Polyethylenterephthalat. The marrow cells are passed sequentially through the two filters and the MK-rich retained populations are collected from the top of the filters. Following removal of excess buffer, a QC Wright-Giemsa-stained cytospin provides a visual appreciation of cells collected (Fig. 1). The remaining MKs are prepared for flow cytometry or EM. This filtration procedure allows the preferential retention of MKs and, on average, the procedure only lasts 15-20 minutes. For electron microscopy, the MKs are being fixed in glutaraldehyde in suspension until further processing. Results 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 based on their large size, expression of platelet-associated antigens and DNA ploidy levels. A quantitative analysis of MK ploidy has been completed in marrow aspirates from 52 patients with hematologic conditions that variably affect the marrow (normal, reactive, dysplastic, and proliferative processes). The number of MK/sample ranged from 134 to 15,266 (mean = 3,253). To determine changes in ploidy levels, the weighted mean of the total DNA content of MKs was calculated in each case. Despite the heterogeneity and low sample size, we noted a significant decrease in ploidy level among marrows from patients with high grade myelodysplasia (MDS) or acute myeloblastic leukemia (AML), indicating a relative increase in the frequency of low ploidy cells in these cases. Morphologically, the corresponding marrows exhibited numerous MK with dysmegakaryopoietic features, such as micromegakaryocytes and a reduced number of mature forms. To determine if the increased number of small, hypodiploid MK were the result of increased proliferation or a maturations arrest, a dual IHC stain was performed on all MDS/blasts/AML cases using CD61 staining combined with the proliferation marker Ki-67. While the background marrow elements demonstrated a high proliferative rate, the MK (identified by CD61) showed no Ki-67 labeling, consistent with an arrest in maturation of these cells. Importance of findings We continue to analyze the DNA content (ploidy level) of MK to determine the quantity and distribution of the various G1 peaks that reflect nuclear lobe partitions in these cells. The demonstration of increased MK that are arrested in maturation in MDS and AML follows other cell lineages that undergo similar changes leading to severe cytopenias. Our data suggest that this analysis may be of diagnostic value in a disease like MDS where hematopathologists face serious diagnostic challenges. This rapid analysis performed on unfractionated and minimally manipulated bone marrow aspirates could potentially be adapted for the routine use in the clinical laboratory.

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 对于MK富集，用缓冲溶液（含8% BSA的HBSS）稀释来自各种血液病患者的废弃骨髓穿刺液，并在微滤前首先使细胞悬液通过100 μ m过滤器。 对于微量过滤，我们最初使用8 - 90 mm圆形尼龙网过滤器（Repligen），但由于该过滤器停产，我们改用2级过滤，其中10 m和5 m网来自Pluriselect（德国），由聚乙烯醇制成。使骨髓细胞依次通过两个过滤器，并从过滤器顶部收集富含MK的保留群体。 去除过量缓冲液后，QC瑞氏-姬姆萨染色的细胞离心涂片提供了所收集细胞的视觉评价（图1）。 制备剩余MK用于流式细胞术或EM。 该过滤程序允许优先保留MK，并且该程序平均仅持续15-20分钟。对于电子显微镜检查，将MK在戊二醛悬浮液中固定，直至进一步处理。 结果 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。 通过流式细胞术基于其大的尺寸、血小板相关抗原的表达和DNA倍体水平鉴定MK。 MK倍性的定量分析已经完成了52例患者的骨髓穿刺血液条件，影响骨髓（正常，反应，发育不良，增殖过程）。 MK/样本数量范围为134 - 15，266（平均值= 3，253）。 为了确定倍性水平的变化，在每种情况下计算MK的总DNA含量的加权平均值。 尽管存在异质性和低样本量，我们注意到高度骨髓增生异常（MDS）或急性髓细胞白血病（AML）患者的骨髓中倍性水平显著降低，表明这些病例中低倍性细胞的频率相对增加。 在形态学上，相应的骨髓表现出大量的MK与dysmegakaryogenetic功能，如小巨核细胞和数量减少的成熟形式。 为了确定小亚二倍体MK数量增加是否是增殖增加或成熟停滞的结果，使用CD 61染色结合增殖标志物Ki-67对所有MDS/原始细胞/AML病例进行双重IHC染色。 虽然背景骨髓成分显示出高增殖率，但MK（通过CD 61鉴定）未显示Ki-67标记，这与这些细胞成熟停滞一致。 调查结果的重要性 我们继续分析MK的DNA含量（倍性水平），以确定反映这些细胞核叶分区的各种G1峰的数量和分布。 在MDS和AML中成熟停滞的MK增加的证明遵循经历导致严重血细胞减少的类似变化的其他细胞谱系。 我们的数据表明，这种分析可能是诊断价值的疾病，如MDS的血液病理学家面临着严重的诊断挑战。 这种对未分级和最低限度操作的骨髓穿刺液进行的快速分析可能适用于临床实验室的常规使用。