DOES A SMALL VOLUME ASPIRATE HAVE MORE CFU-F THAN A LARGER DRAW THAT IS THEN VOLUME REDUCED BY CENTRIFUGATION?

In order to centrifuge marrow, you need to add 10% by volume anti-coagulant. This dilutes the end product. You do not need to add that volume of anti-coagulant if you are not centrifuging.

When you centrifuge marrow, approximately 40% of the stem cells are discarded with the red cells. Stem cells / progenitor cells when they are cycling build up nucleic mass prior to division. At this stage their density increases dramatically to the point where they have a similar density to a red cell. Centrifugation protocols capture a range of cells based on density. In any given aspirate, approximately 40% of the stem cells have density outside what is typically captured in the buffy coat.

Typically, the centrifugation protocol volume reduces the aspirate from 60cc to 10cc. This 10cc is then used to hydrate the substrate. However, in order to reverse the effect of the anti-coagualnt, to make the marrow clot, about 1cc of calcium chloride and thrombin is mixed with the concentrate; another 10% dilution.

A centrifuge does not distinguish between nucleated cell from marrow verses blood. So a sample may have a lot of nucleated cells but very few came from marrow. A CFU-f test is often performed on marrow aspirate because CFU-f are found in marrow but not blood.

Finally, there is no way to draw 60cc of marrow, no matter how careful, and not get significant peripheral blood dilution. A careful draw of 60cc with multiple punctures typically, at best, gets on average 300 cfu-f per mL.

The math play for a typical centrifuged marrow aspirate sample plays out as follows:

CFU-f per mL 300
volume 60 mL
Total CFU-f per mL 18,000
Recovery in the concentrate 60%
Total CFU-f in Concentrate 10,800
Concentrate Volume 7 mL
CFU-f per mL 1,543
HSS / Hedge Study 1,270
This compares to 2,000 CFU-f per mL drawn from a single site in the marrow.

Juopperi TA, Schuler W, Yuan X, Collector MI, Dang CV, Sharkis SJ. Isolation of bone marrow-derived stem cells using density-gradient separation. Experimental hematology 2007;35:335-41.

Bhartiya D, Shaikh A, Nagvenkar P, et al. Very small embryonic-like stem cells with maximum regenerative potential get discarded during cord blood banking and bone marrow processing for autologous stem cell therapy. Stem cells and development 2012;21:1-6.

Vishal Hegde MD et al; “Title: A prospective comparison of three approved systems for autologous bone marrow concentration demonstrated non-equivalency in progenitor cell number and concentration.” Journal of Orthopaedic Trauma Publish Ahead of Print

MUSCHLER G, et al “Aspiration to Obtain Osteoblast Progenitor Cells from Human Bone Marrow: The Influence of Aspiration Volume” The Journal of Bone and Joint Surgery; VOL. 79-A, NO. 11. Cleveland Clinic

Hernigou. P et al “Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells” Int Orthop 2013 Nov;37 (11): 2279-87