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 morphological 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, last year, we entered a collaborative effort with professionals in the Department of Pathology of the Walter Reed National Military Medical Center (WRNMMC) who have access to marrow samples, in order to obtain adequate and representative material for our studies. This process took a long time and although we received some samples from that institution, the amount and/or quality of the samples was sub-optimal and, in most instances, did not yield a sufficient number of megakaryocytes for appropriate analysis. We have recently decided to terminate the acquisition of samples for this purpose and analyze the results obtained with the samples already received. We are analyzing the DNA content (ploidy level) of megakaryocytes in these samples in order to determine the quantity and distribution of the various G1 peaks that reflect nuclear lobe partitions in these cells, as well as the expression of platelet-associated glycoproteins that may be altered in hematologic disorders with morphological MK abnormalities. We are still collecting selected cases of marrow disorders that affect megakaryocytes for appropriate characterization of changes and comparisons between normal, reactive, dysplastic and proliferative processes. 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
