Rapid and Simple Isolation and Concentration Procedure for Human Megakaryocytes

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Abstract

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.