Abstract
Numerical and morphologic abnormalities of megakaryocytes (MKs) are present in a variety of primary or secondary bone marrow (BM) disorders such as myeloproliferative neoplasms, myelodysplastic syndromes or ITP. Currently, these changes are assessed exclusively on microscopic preparations of marrow aspirates and biopsies and are used as criteria for disease diagnosis, classification and therapy monitoring despite the inherent subjectivity of microscopic evaluations. In contrast to other cells, adequate studies of freshly isolated MKs have proven difficult because of the relative rarity of these cells in the BM (∼0.05% of total nucleated cells) and separation techniques such as density gradients, magnetic beads, centrifugal elutriation or fluorescence activated cell sorting are labor-intensive, time-consuming or costly. The primary means for studying MKs is based on the isolation of progenitors primed with cytokines to differentiate in culture. While extremely valuable, these techniques are not directly translatable to the routine clinical arena for assessing MK pathology in human BM. Mature marrow MKs are large, polyploid cells whose size distribution overlaps minimally with that of all other marrow cells. This distinct size threshold is a discriminatory parameter for MK isolation. Thus, we developed a simple and inexpensive manual mesh filtration method for separation of MKs that allows a rapid and easy, size-based concentration and purification of these cells.
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 heparin/albumin-containing buffered saline solution. The cell suspensions were first filtered through a 70µm filter to remove large BM stromal particles and then twice (by gravity) through 9mm-diameter wetted 8 µm nylon filters placed in a simple plastic holder. This filtration procedure allowed the retention of MKs and removal of smaller cells. The MK-rich suspension was collected by rinsing and flushing the filters with the buffered solution. On average, the procedure only lasted 15 minutes. The pre-filtered, 8 µm filter-retained, and effluent cell suspensions collected were stained with an FITC-anti CD61 or PE-anti CD41a antibody [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. No cell lysing, fixation or centrifugation was used at any step of the MK separation procedure.
The median (range) volume of the BM aspirate 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 simple, gentle, 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 this 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 require adequate cell representation and purity.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
