The science behind our services

Clinical MSC enumeration by flow cytometry.

How we harvest bone marrow, concentrate it into BMAC, and count the cells it contains. We measure each step.

Jason Glowney, MD · Autologous Orthobiologics

This is a detailed subject, and what it means for your situation depends on your history and your imaging. Dr. Glowney is glad to work through it with you.

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Step 01

Harvest

Multi-site, low-volume aspiration from the posterior iliac crest.

Step 02

Concentrate

Controlled centrifugation that protects viability.

Step 03

Enumerate

Count, viability, and MSC-enriched populations by flow cytometry.

Step 04

Deliver

Same-day, same-visit, with the data behind it.

In short

The quality of the final concentrate depends on every step together: how the marrow is drawn, how hard it is spun, and whether the cells are checked afterward. We measure each one.

01 · Clinical & scientific rationale

Why we measure each step.

Treatments made from bone marrow depend on how the marrow is drawn, how it is processed, and how the cells are checked. The cells of most interest, mesenchymal stromal cells (MSCs), are rare. They make up roughly 0.001–0.01% of the cells with a nucleus in bone marrow, and how many we recover depends heavily on technique.1,2,3

Our approach integrates optimized bone marrow aspiration, controlled preparation of bone marrow aspirate concentrate (BMAC), and quantitative flow cytometric analysis using the Sysmex XF-1600 platform to characterize total nucleated cells, viability, and MSC-enriched subpopulations in live, freshly processed samples.

This page outlines our end-to-end workflow, from BMA harvest to BMAC concentration and MSC enumeration, and illustrates how these steps combine to support evidence-informed autologous orthobiologic procedures.

02 · Harvest technique

How we harvest bone marrow.

Bone marrow aspirate is obtained from the posterior superior iliac spine (PSIS) using a multi-site, low-volume aspiration strategy. Rather than drawing large volumes from a single marrow cavity, the needle is redirected between small-volume pulls (approximately 5 mL per draw) to reduce peripheral blood dilution and enrich mononuclear cell fractions with colony-forming unit-fibroblast (CFU-F) potential.

This technique is supported by work showing that drawing marrow in small volumes with a small syringe gives a higher concentration of progenitor cells than larger draws.6

Following aspiration, the marrow is passed through a micron-scale filter to remove bone fragments and clots while preserving cellular integrity. A defined portion of the filtered aspirate may be retained unprocessed, while the majority proceeds to controlled concentration.

Why small-volume, multi-site

Large single-site draws pull in more peripheral blood and dilute the progenitor fraction. Many small pulls, with the needle moved between draws, recover more progenitor cells.

03 · Concentration

How we concentrate it.

Bone marrow aspirate concentrate is generated using centrifugation parameters selected to balance effective mononuclear cell enrichment with cell-viability preservation. Spinning the marrow too hard can raise the apparent cell count but damage cell membranes and lower the share of living cells.

Our processing protocols emphasize:

Moderate centrifugal forces
Preservation of mononuclear cell populations
Minimization of mechanical stress
Maintenance of sterility throughout processing

The result is a BMAC product enriched for mononuclear cells while maintaining high overall viability, consistent with published work showing that harvest and processing technique affect how many MSCs end up in the final concentrate.2

A residual (RES) fraction, the post-collection material remaining in sterile tubes following buffy-coat recovery, is also evaluated. It shows how well cells were recovered into the final BMAC.

04 · Enumeration

How we count and characterize cells.

Platform

Sysmex XF-1600 flow cytometer

We analyze live bone marrow samples before and after concentration, sample by sample, instead of relying on the kit maker's specifications.

Nucleated cell count Viability MSC-enriched %

Nucleated-cell counting & viability

To quantify nucleated cells, aliquots of BMA, BMAC, and RES samples are diluted in buffered solution and treated with a lytic reagent that removes cytoplasmic membranes while preserving nuclei. Forward-scatter-based size gating isolates nuclei within a defined micrometer range, allowing accurate calculation of nucleated-cell concentrations.

Viability is assessed using DNA-binding viability dyes that selectively enter cells with compromised membranes. Viable and non-viable populations are distinguished by fluorescence intensity, enabling a percent-viability calculation for each sample fraction. In the example shown below, viability was high in both BMA and BMAC samples.

Identifying MSC-enriched populations in fresh samples

Unlike culture-expanded cells, freshly isolated bone marrow contains a heterogeneous mixture of hematopoietic, stromal, and progenitor populations. In this context, MSCs are best identified indirectly, using flow-cytometric marker combinations that correlate with CFU-F activity rather than relying on in-vitro expansion phenotypes.

CD45 −Lineage exclusion

Negative selection removes hematopoietic-lineage cells, focusing the analysis on the non-hematopoietic stromal fraction.

CD271 (LNGFR) +Positive selection

Correlates strongly with clonogenic MSC potential in ex-vivo marrow samples.4

TNAP / MSCA-1 +Positive selection

Tissue non-specific alkaline phosphatase; a further marker associated with CFU-F-forming MSCs in fresh marrow.5

CD73 · CD90 · CD105Not used here

Commonly associated with culture-expanded MSCs, but often downregulated or absent in freshly isolated marrow, so they are not relied upon for primary enumeration.

Using this approach, the Sysmex XF-1600 enables direct comparison of MSC-enriched fractions from native BMA, processed BMAC, and residual layers, providing an internal quality-control metric for concentration efficiency.

05 · Representative analysis

Figures & comparison.

The figures below show representative flow-cytometric analyses of an example patient sample on the Sysmex XF-1600. They demonstrate methodology and comparative enrichment, not fixed dosing thresholds.

Figure 1. Native bone marrow aspirate (BMA). CD73 on the vertical (FITC) axis, CD271 on the horizontal (APC) axis; the upper-right gate marks lineage-negative, CD73⁺/CD271⁺ events consistent with MSC-enriched populations in fresh marrow.

Figure 2. Bone marrow aspirate concentrate (BMAC) after controlled centrifugation. The gated CD73⁺/CD271⁺ region shows relative enrichment versus native BMA, with high viability maintained.

Figure 3. Residual (RES) fraction remaining in sterile tubes after buffy-coat collection. Markedly fewer CD73⁺/CD271⁺ events than BMAC, confirming effective recovery into the final concentrate.

Figure 4. Quantitative comparison of BMA, BMAC, and RES from live-sample analysis on the Sysmex XF-1600, reflecting nucleated-cell concentration, viability, and MSC-enriched population metrics.

Figures show representative data from a single example sample.

Together, the figures show four points:

MSCs are a very small fraction of total nucleated cells in native marrow, typically well below 1%, consistent with prior CFU-F and flow-cytometry studies.
Controlled BMAC processing increases the relative frequency of MSC-enriched populations despite modest changes in absolute nucleated-cell concentration.
The residual fraction contains substantially fewer MSC-associated events, confirming effective recovery into the final BMAC product, an internal process-control metric.
High viability is preserved across all fractions, underscoring the role of aspiration technique and moderate centrifugation in maintaining cellular integrity.

Careful processing can concentrate these cells without damaging them, and counting them means we don't have to guess at the dose.2

06 · What it means clinically

Clinical implications.

Counting cells and MSC-enriched populations lets us report what each preparation actually contains. By integrating aspiration technique, controlled processing, and validated flow-cytometric analysis, we are able to:

Confirm cell viability and concentration
Quantify the enrichment achieved through BMAC processing
Avoid unsupported assumptions about cellular dose
Base our records on measured values

Published calls for standard reporting of cell therapies ask for this kind of measurement.

07 · Regulatory framework

Same-day BMAC vs. culture-expanded workflows.

We keep same-day BMAC, which is used in patient care, separate from cells grown in culture, which are used only for research.

Clinical use

Same-day BMA / BMAC

Autologous bone marrow aspirate, processed and administered within a single procedural encounter. Designed to align with Section 361 of the Public Health Service Act and 21 CFR Part 1271, including:

Autologous use
Minimal manipulation (centrifugation and filtration only)
Homologous use within musculoskeletal tissues
No cell expansion, genetic modification, or prolonged ex-vivo culture

BMAC is a concentrated mix of cells and signaling molecules from your own marrow: mononuclear cells, blood-forming progenitor cells, stromal cells, and platelets. It works through signaling that can support the body's own repair. It does not replace tissue.

Research & characterization

Culture-expanded workflows

Short-term culture and cell expansion are conducted outside same-day clinical care, used exclusively for research, characterization, and quality evaluation under controlled laboratory conditions.

Expanded populations may meet internationally recognized immunophenotypic criteria for MSCs (e.g. CD73⁺/CD90⁺/CD105⁺), but differ fundamentally from fresh marrow in:

Cellular homogeneity
Surface-marker expression
Proliferative capacity
Regulatory classification

Culture-expanded cells are not administered clinically under same-day protocols and are clearly segregated from patient treatment pathways.

Regulatory note

Surface markers commonly associated with culture-expanded MSCs are often downregulated or absent in native marrow populations and should not be used to infer cell identity or dose in same-day BMAC procedures.

Summary

One interconnected workflow.

Each step, from harvest to counting, affects the next, so we measure all of them. Outcomes depend on the cumulative effect of aspiration technique, processing forces, and objective cellular validation.

Using the Sysmex XF-1600 and evidence-based protocols, Boulder Biologics applies a quantitative, transparent framework for autologous orthobiologic care that reflects current scientific understanding and regulatory requirements.

References

Sources cited.

Jones EA, Kinsey SE, English A, et al. Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells. Arthritis Rheum. 2002;46(12):3349–3360.
Jeyaraman M, Bingi SK, Muthu S, et al. Impact of the process variables on the yield of mesenchymal stromal cells from bone marrow aspirate concentrate. Bioengineering (Basel). 2022;9(2):57.
Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologous bone-marrow grafting for nonunions: influence of the number and concentration of progenitor cells. J Bone Joint Surg Am. 2005;87(7):1430–1437.
Quirici N, Soligo D, Bossolasco P, et al. Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies. Exp Hematol. 2002;30(7):783–791.
Sobiesiak M, Sivasubramaniyan K, Hermann C, et al. The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase. Stem Cells Dev. 2010;19(5):669–677.
Hernigou P, Homma Y, Flouzat Lachaniette CH, et al. Benefits of small volume and small syringe for bone marrow aspirations of mesenchymal stem cells. Int Orthop. 2013;37(11):2279–2287.