Selective enrichment of rare bone marrow cell populations for electron microscopy

Transmission electron microscopy (TEM) is a unique high-resolution method allowing to study the cell ultrastructure of normal and abnormal cells. One of the factors hindering wider application of TEM for diagnosis is the challenges associated with the collection of a sample that would be both enriched in cells of interest and suitable for TEM. The aim of this study was to develop a method for the purification of megakaryocytes from a bone marrow aspirate using antibodies to megakaryocyte surface antigens immobilized on slides as well as to describe a protocol for preparing such isolated cells for a TEM analysis. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Ministry of Healthcare of the Russian Federation. For megakaryocyte purification, monafram (F(ab')2 – a fragment of a murine monoclonal antibody to glycoprotein IIb–IIIa) was adsorbed on a glass slide modified with dimethyldichlorosilane. A suspension of mononuclear cells purified from the bone marrow aspirate using the Histopaque 1077 gradient was incubated with the immobilized antibodies for 2 hours at 4°С with mixing every 20 min. The sample was then washed to remove nonspecifically bound cells, fixed with 2.5% glutaraldehyde, postfixed with 1% osmium tetroxide in water, consecutively dehydrated in 30, 50, 70, 90 and 100% acetone and embedded in a thin 0.3–0.5 mm layer of Epon 812 mixed with acetone at 1:2 and 2:1 ratios. After the polymerization of the first thin Epon 812 layer, a cylinder 8 mm in diameter and 10 mm in height was glued on top of the region with bound cells and was left to polymerize. The polymerized resin was then detached from the glass slide using a scalpel, cut using an ultramicrotome and analyzed using TEM. Using this protocol, we studied bone marrow aspirates of 3 patients with essential thrombocythemia. The donors, patients and/or their legal representatives gave consent to bone marrow aspiration and further biomedical research. The obtained electron photomicrographs show all the characteristic features of megakaryocytes including loose nucleus, granules and cisternae of the demarcation membrane system and are in agreement with corresponding images in the existing literature. The suggested protocol allows to obtain TEM samples enriched in rare blood or bone marrow cells using significantly less time and money on sample preparation and photomicrography. This approach is universal and can be used not only for megakaryocytes but for other cells as well, including erythroid precursors.