Rapid and Simple Isolation and Concentration Procedure for Human Megakaryocytes

背景 巨核细胞(MK)是骨髓中一种罕见的细胞群，估计占有核细胞总数的0.1-0.5%。MK的数量和形态异常在各种原发和继发性骨髓疾病中都有描述，但这些细胞的稀缺性使分析变得困难，后续细胞分析的浓缩技术复杂且劳动强度大。目前，MK的变化仅在显微镜下进行评估(骨髓抽吸物和活检)，并被用作疾病诊断、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。与其他造血细胞或淋巴样细胞相比，由于这些细胞的相对稀有，对新鲜分离的MK进行充分的定量研究被证明是困难的。即使是非常适合于快速检测罕见细胞群的流式细胞术(FCM)，在评估骨髓MK时也面临着严重的困难，而且这些细胞的分析不是临床实验室骨髓细胞常规诊断工作的一部分。可以使用分离技术分离MK，如密度梯度、磁珠、离心洗脱或荧光激活细胞分选，但这些方法劳动密集、耗时或成本高昂。成熟的骨髓MK是大的、多倍体的细胞，研究表明，它们的大小分布与所有其他骨髓细胞的大小重叠最小。这种不同的大小阈值意味着，大小本身就是MK分离的一个歧视性参数。因此，我们开发了一种简单且廉价的人工网目过滤方法来分离MK，这种方法允许快速和容易地基于大小浓缩和纯化这些细胞。 材料和方法 我们检查了15个适合我们分析的废弃的匿名骨髓抽吸物样本。这些样本来自最初提交给我们实验室进行评估的各种血液病患者。样本年龄1-4天，平均1.5天。样品用缓冲的生理盐水稀释，细胞悬液通过适当大小的筛网过滤。这种过滤程序允许优先保留MK，平均只持续15分钟。细胞悬液用FITC-抗CD61或PE-抗CD41a抗体Becton Dickinson(BD)标记MK，同时用细胞通透性DNA结合DRAQ5(Cell Signal Technology，细胞信号传递技术)鉴定所有有核细胞。用C12Resazurin(分子探针)检测细胞活性。采用CANTO II流式细胞仪(BD)进行细胞分析。使用流式细胞仪绝对计数标准珠(BANGS)进行绝对MK计数。用流式细胞仪测定所有有核细胞的巨核细胞百分率来表示巨核细胞集落形成效率。 结果和结论 本研究中使用的骨髓样本的中位数(范围)为0.54(0.24-1.99)毫升。流式细胞仪根据MKs的大小、血小板相关抗原的表达和DNA倍体水平对其进行鉴定。MK回收率的中位数(范围)是原始MK数量的31(14-100)%，每毫升BM吸出物的产量为9882(1,519-49,921)MK。过滤后MK在所有有核细胞中的中位数(范围)为39(14-68)%，是MK浓缩的904(439-3029)倍。滤过后骨髓间充质干细胞活性接近100%。这种快速、廉价的分离和浓缩方法可使MK的回收和提纯达到或优于其他更精细的技术。尽管所使用的样本具有内在的异质性，但我们获得了相当好的每毫升骨髓抽吸物中巨噬细胞集落刺激因子的回收率和900多倍的中位巨噬细胞集落刺激因子浓度。新分离的巨核细胞的产量和纯化水平可用于各种原发或继发性骨髓疾病的研究。特别是，它应该促进分析方法的应用，如流式细胞术或原位杂交，甚至对需要足够的细胞代表性和纯度的生化或分子测试有用。 当前研究进展 利用流式细胞术，我们目前正在检测从新鲜抽吸物中分离的MK细胞的DNA含量(倍体)和各种表面抗原的表达，如上所述，从正常志愿者和各种血液病患者的骨髓中分离出的MK细胞。这项研究的目的是建立MK倍体和/或免疫表型的改变，如果有的话，在那些基于形态学原因被认为MK异常的疾病中，例如骨髓增生异常或骨髓增生性肿瘤。不幸的是，目前美国国立卫生研究院这些疾病的发病率很低。此外，在这家机构中，患有骨髓疾病的血液病患者需要进行广泛的研究和诊断测试，这需要大量的骨髓样本，通常留给我们研究的材料相对较少。为此，我们联系了沃尔特里德国家军事医学中心病理科的专业人员，他们可以获得骨髓样本，以建立一个合作努力，为我们的研究获得足够的和有代表性的材料。 摘要：劳尔·C·布莱兰，伊莱恩·凯·乔丹。从骨髓抽吸物中快速分离和浓缩人巨核细胞血液2013 122：5269

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