Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

This method can help to answer key questions in the diagnosis of myeloid diseases such as myelodysplasia syndromes or other hematological diseases that evolve with maturation abnormalities in myeloid cell lineages. The main advantage of this technique is that it reduce the subjectivity of investigator in that analysis and interpretation. This method can provide insight into which are the most discriminatory parameters for myelodysplasia.

It allows quantification of the differences from normal myeloid cell populations. Demonstrating the procedure will be Tiphanie Picot, a post-doctoral fellow from our laboratory. Begin by mixing 600 microliters of the primary cell sample with 10 milliliters of washing buffer in a 15 milliliter conical tube for two centrifugations and 10 milliliters of fresh washing buffer per wash.

After the second wash, re-suspend the pellet in 400 microlitres of fresh washing buffer, and transfer 350 microliters of the cell suspension into a five milliliter polypropylene FACS tube, containing the entire panel of backbone antibodies. After thoroughly mixing the cells, pipette equal volumes of the cell antibody solution into three new polypropylene tubes, and bring the final volume in each tube up to 200 microliters with fresh washing buffer as necessary. Next, add the appropriate volume of antibodies against the cell surface markers of interest with mixing, for a 30-minute incubation at room temperature, protected from light.

At the end of the incubation, add two milliliters of lysing solution with mixing, for a 10-minute incubation at room temperature, protected from light. Collect the lysed cells by centrifugation, and discard all but the last 50 microliters of supernatant in each tube, without disturbing the pellet. Re-suspend the pellets in the remaining supernatant, with gentle mixing, and add two milliliters of fresh washing buffer to each sample.

After the second wash, discard the supernatant, leaving approximately 50 microliters residual volume in each tube, without disturbing the pellet. Re-suspend the pellet in 200 microliters of PBS with mixing, for immediate analysis. To read the samples on a flow cytometer, first open a new experiment in the flow cytometer software, and rename the experiment according to the name, type of sample, and date.

Create a new specimen for three tubes, and specify the antibodies used in each tube in the experiment layout. Open the cytometer settings, and select application settings, to apply the values obtained in the monthly setup. Open a new global worksheet and create the appropriate dot plots for gating the singlet cells, as well as for the granulocyte, monocyte, blast, and lymphocyte populations.

Create a new global worksheet for the compensation controls. Then, acquire 500, 000 events from each tube, at a medium acquisition rate, exporting the data as fcs 3.0 files, after their technical validation. Before starting a new analysis, download the cyt files containing the normal bone marrow data, for neutrophil, monocytic, and nucleated red lineages, and the analysis files in inp format.

Then, save the data onto your desktop. In order to save the neutrophil maturation to the data base, first open the Infinicyt software and then open the cyt file corresponding to the neutrophil maturation data. Draw the maturation pathway on the APS graph, and save the neutrophil's maturation pathway to the database.

Open the fcs file for the neutrophil lineage maturation that has to be analyzed. Upload the neutrophil maturation inp file from the profiles tab. The analysis for this lineage is divided in three steps.

Start with selection of CD34 positive, neutrophil-committed blasts. To this aim, use an intersection of seven gates, to allow selection of the CD34 positive, CD117 positive, HLADR low, CD10 negative, CD13 positive, CD11 b-negative events. Continue the analysis with the selection of the CD117 positive, CD34 negative neutrophil precursors.

Continue the analysis with the selection of more mature neutrophil cells. Next, show the analysis of monocytic lineage cells. Draw the maturation pathway on the APS graph.

Verify that the arrow indicating the sense of maturation is oriented from immature, monocytic cells, CD14 negative, IREM2 negative, to the mature monocytes, CD14 positive, IREM2 positive. Save the maturation pathway for monocytic lineage to the database. Open the fcs file for monocytic lineage maturation that has to be analyzed.

Upload the profile for monocytic lineage analysis. To identify the monocytic lineage cells, use an intersection of four gates that allows selection of CD64 positive high, CD117 positive, CD117 negative, HLADR positive events. Assign these events to the monocytic tab in the population hierarchy tree.

Open the NRC cyt file that has to be saved in the NRC database. Draw the maturation pathway on the APS graph, and save the nucleated red cells maturation pathway to the database. Open the fcs file that has to be analyzed.

Upload from the profiles the template of analysis for nucleated red cells. The analysis for this lineage is divided in two steps. Starting with the selection of the CD34 positive, erythroid committed blasts.

Here, use an intersection of seven gates to allow selection of the CD34 positive, CD117 positive, HLADR low, CD105 positive, CD33 negative, CD36 positive, CD71 positive events. Assign these events to the NRC tab in the population hierarchy. Remove the CD34 positive erythroid committed blasts from visibility.

To identify more mature erythroid cells, use an intersection of four gates that allow discrimination of CD45 negative and CD45 positive low, side-scatter low, CD36 positive high, and CD71 positive high. Then, remove the CD36 high side scatter low platelets from the nucleated red cells, and assign this population to the NRC tab. To assess myeloid maturation in the bone marrow compartment, open the cyt file corresponding to the lineage of interest from the case that has to be evaluated.

Keep only the events assigned to the neutrophil's tab in the population hierarchy tree visible. Draw the maturation pathway on the APS graph and load the database corresponding to the neutrophil's maturation in normal bone marrow, and compare data. If the data are at least partially compatible, the software will create a normalized maturation differences diagram, and a parameter band, to visualize the maturation differences.

To compare the monocytes maturation in the bone marrow compartment, for a new case with data from normal bone marrows, including in the monocytes database, open the cyt file corresponding to the monocytic cell lineage. Keep only the events assigned to the monocytes tab in the populations hierarchy tree visible, and draw the maturation pathway on the APS graph. Load the database corresponding to the monocytes maturation in normal bone marrows, and compare data.

To compare the nucleated red cells maturation in the bone marrow compartment, for a new case, with data from the normal bone marrows, included in the NRC database, open the cyt file corresponding to the NRC's lineage. Keep only the events assigned to the NRC tab in the population hierarchy tree visible. And draw the maturation pathway on the APS graph.

Load the database corresponding to the NRC maturation in normal bone marrows, and compare data. Then, to visualize the significance of the differences between the new file and the data included in the database, click normalized maturation differences and zoom. Remember that methods that use the hierarchical classification algorithms in flow cytometric data analysis are highly subjective and inherently inaccurate, because they do not count for cell population overlap.

The evaluation of new cases of cytopenia, suspected of being MDS against the myeloid normal maturation databases, allow for the identification of abnormally-expressed maturation antigens of neutrophil, monocyte, and NRC lineages, even in cases without cytological or cytogenic abnormalities. While attempting this procedure, it's important to remember to acquire the flow cytometric data, in standardized manner, to perform monthly cytometer setting setup, daily checks, to pipette rigorously the antibody before use, and to respect the staining times. Following this procedure, another methods like compass, can be performed to compare the different group of cases, and to answer to additional questions, such as what is the final typic imprint for a particular group of cases.

After its development, this technique paved the way for the researchers interested in exploring myeloid cell maturation patterns, in cases of clonal hematopoiesis of indeterminate potential to identify the final typic changes reliably related for dysplastic process. Please note that updating the database with increased number of data from healthy donors may improve the robustness of the analysis.