The overall goal of this procedure is to estimate leukemia stem cells in acute myeloid leukemia and in patient-derived-xenografts by flow cytometry at diagnosis and at treatment follow up. This method can answer key questions in the hematology/oncology field, such as the clinical relevance of the quantitative assessment of leukemia stem cells by flow cytometry in acute myeloid leukemia. The main advantage of this technique is that it is applicable to most hematology/oncology laboratories with commonly used antibodies.
This procedure will be demonstrated by Thomas, a post-doc, Fanny, a grad student, and Adeline, an engineer from my laboratory. To begin, collect two milliliters of bone marrow from de novo AML in tubes containing 1.8 milligrams per milliliter of the anti-coagulant EDTA. Isolate mononuclear cells from red blood cells and granulocytes by first diluting bone marrow in three volumes of PBS.
Then, carefully overlay one volume of ficol with one volume of diluted bone marrow. Spin the density gradient at 300g for 30 minutes without a break. Then, transfer the white buffy coat layer of mononucleated cells into a new sterile tube.
To remove the supernatants contaminating serum components, add 10 milliliters of PBS and centrifuge the cells at 300g for five minutes before repeating the wash. Lyse the red blood cells by adding two milliliters of lysis buffer to the cell pellet and gently vortex. Then, incubate the cells at room temperature for five minutes.
Centrifuge the cells at 300g for five minutes and remove the supernatant. Then, use PBS to wash the cells. After isolating blast cells from bone marrow according to the text protocol, under a laminar flow hood, insert an unconditioned NSG mouse into a restraining device and inject five times 10 to the sixth AML blast cells into the tail vein.
Return the mouse to an individual ventilated cage under conventional and specific pathogen-free conditions. Every two weeks, use a submandibular collection method with a hematocrit capillary to draw 60 microliters of blood. Transfer the capillary into a five milliliter tube then with a sterile gauze pad, apply gentle pressure to stop the bleeding.
Add one milliliter of lysis buffer to the tube and gently vortex. Then, incubate the cells at room temperature for five minutes. Centrifuge the cells at 300g for five minutes.
Then, after removing the supernatant, add 100 microliters of PBS with 10%BSA, three microliters of anti-human CD45 FITC, incubate for 30 minutes. Then, wash the cell pellet two times in PBS, two milliliters, with 10%BSA and centrifuge at 300g for five minutes. Every two weeks, use flow cytometry to analyze the engraftment by chimerism analysis of human versus murine CD45 expression.
When the blast count is greater than 70%or severe clinical signs of sickness are observed, sacrifice the animal and collect mononuclear blast cells from a crushed spleen and from bone marrow by using PBS to flush tibias and femurs as previously described. To carry out panel staining, combine conjugated antibodies with human primary or patient-derived-xenograft, or PDX samples, and vortex the tubes. Then, incubate the cells in the dark at room temperature for 15 to 30 minutes.
Remove the unbound antibodies by using two milliliters of PBS to wash the cells. Re-suspend the cells in 450 microliters of PBS, then proceed to flow cytometry analysis and MRD monitoring. To accurately and reproducibly assess membrane of cytoplasmic markers by flow cytometry, verify optical alignment.
Proper tuning of the 3D system and optical sensitivity and stomatization using stomazine calibration beats every day. Use normal bone marrow to set the gates for CD38. A minimum of 500, 000 events are required and it is verified that each flow run is regular and homogenous.
In addition, doublets, as well as dead cells and cell debris are eliminated. Immature hematopoietic cells are defined by being CD45 intermediate, SSC low. And the CD38 positive control or hematogones, are CD19/CD38 double positive.
Then P8 or CD38 positive, P7 or CD38 intermediate, and P6 or CD38 negative are defined. The P6 cells can be further subdivided into HSC, MPP, and LSC. Since it is a normal bone marrow it is LSC negative.
Using multiparametric flow cytometry, it was found that the CD34 positive, CD38 negative blast fraction was higher in PDX compared to the diagnostic patient sample. Interestingly, a higher percentage of putative LSC was found in the PDX compared to the primary patient sample, suggesting a possible enrichment of malignant progenitor cells in the hematologic tissues in this mouse model. Upon comparison of the blast progenitor populations in two different patient samples, the CD34 positive, CD38 negative fraction increased from 0.06%at diagnosis to 3.33%at the first treatment follow-up in the first patient.
In contrast, the CD34 positive, CD38 negative fraction decreased in the second patient from 7.8%at diagnosis to 2.27%at the first treatment follow-up. Accordingly, the putative LSC fraction increased in the first patient from 0%at diagnosis to 0.65%and decreased in the second patient from 6.57%at diagnosis to 1.44%at treatment follow-up. Minimal residual disease or MRD monitoring, using as molecular markers the NPM1 mutation in patient one and the CBF-beta MYH11 gene fusion for patient two showed that molecular MRD decreased less in the first patient compared to the second patient for the first treatment follow-up point.
Once mastered, this technique can be done in two hours if it is performed properly. While you're attempting this procedure, it is important to verify flow cytometric settings and CD38 gauging. Following this procedure, additional methods can be performed, like flow cytometric cell sorting in order to answer additional questions like functional leukemia stem cell assess.
After watching this video you should have a good understanding of how to detect and quantify leukemia stem cells in patient samples in patient-derived-xenografts. Don't forget that working with patient samples can be hazardous and precautions such as GOP guidelines and institutional safety procedures should always be taken while you are performing this procedure.
