The science behind our services

Clinical MSC enumeration by flow cytometry.

Autologous bone marrow harvest, BMAC concentration, and objective cellular characterization, the full workflow, and how each step is measured rather than assumed.

Jason Glowney, MD · Autologous Orthobiologics

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

Outcomes depend not on any single step, but on the cumulative effect of aspiration technique, processing forces, and objective cellular validation. We measure each one.

01 · Clinical & scientific rationale

Why we measure, rather than assume.

Autologous bone marrow–derived orthobiologic therapies rely on careful attention to harvest technique, processing methodology, and objective cellular characterization. Mesenchymal stromal cell (MSC)–containing populations are present at very low frequencies within native bone marrow, typically on the order of 0.001–0.01% of total nucleated cells, and their yield is highly sensitive to aspiration technique, processing forces, and operator-dependent variables.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 foundational work demonstrating significantly higher progenitor cell yields with small-volume, multi-pocket aspiration methods than with large-volume single-site draws.3

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, repositioned between draws, recover more of what matters.

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. Excessively high g-forces, while capable of increasing apparent nucleated-cell density, have been shown to compromise cell-membrane integrity and reduce functional viability.

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 observations that processing technique is a significant determinant of final cellular quality.4

A residual (RES) fraction, the post-collection material remaining in sterile tubes following buffy-coat recovery, is also evaluated. It tells us how efficiently cells were distributed and confirms that clinically meaningful populations were retained in the final BMAC product.

04 · Enumeration

How we count and characterize cells.

Platform

Sysmex XF-1600 flow cytometer

Quantitative analysis of live bone marrow samples before and after concentration, objective, sample-by-sample, no reliance on kit 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. Across representative cases, both BMA and BMAC samples demonstrate high viability, reflecting the use of controlled processing conditions.

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.5

TNAP / MSCA-1 +Positive selection

Tissue non-specific alkaline phosphatase; a further marker associated with CFU-F–forming MSCs in fresh marrow.6

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 are representative case data. Drop the corresponding Sysmex exports into each slot.

Read together, the figures illustrate several well-described principles from the orthobiologic literature:

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.

The takeaway: meaningful biological enrichment can be achieved without excessive mechanical stress or reliance on unverified assumptions about cellular dose.2

06 · What it means clinically

Clinical implications.

Objective enumeration of nucleated cells and MSC-enriched populations brings transparency and rigor to autologous orthobiologic procedures. 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
Align clinical practice with contemporary translational research

This reflects the growing consensus that orthobiologic therapies should be characterized and reported using measurable cellular parameters rather than relying solely on kit specifications or procedural assumptions.

07 · Regulatory framework

Same-day BMAC vs. culture-expanded workflows.

Our procedures clearly differentiate same-day bone marrow aspirate concentrate (BMAC) workflows from culture-expanded cellular workflows, consistent with current FDA guidance and established regulatory distinctions.

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, heterogeneous, cell-containing biologic, mononuclear cells, hematopoietic progenitors, stromal cells, platelets, and signaling molecules, intended to support endogenous repair through biological signaling rather than tissue replacement.

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.

Autologous BMA harvest, BMAC concentration, and MSC enumeration are not independent steps, they are one connected workflow. 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, et al. Enumeration and phenotypic characterization of mesenchymal stromal cells in bone marrow. 2002.
Kraeutler MJ, et al. Bone marrow aspirate concentrate and the yield and frequency of mesenchymal stromal cells. 2021.
Hernigou P, et al. Percentage of progenitor cells with concentration of bone marrow: small-volume, multi-site aspiration. J Bone Joint Surg Am. 2005. (See also Hernigou et al., 2025.)
Bansal H, et al. Processing technique as a determinant of cellular quality in bone marrow concentrate. Scientific Reports. 2021.
Quirici N, et al. Isolation of bone marrow mesenchymal stem cells by anti–nerve growth factor receptor (CD271) antibodies. Experimental Hematology. 2002.
Sobiesiak M, et al. The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase. Stem Cells and Development. 2010.