Myeloma Blood CTCs: Why Single-Platform and NGF Counts Differ

Why better detection does not guarantee equivalent enumeration

Circulating tumour plasma cells (CTCs) can be enumerated in peripheral blood by flow cytometry, but the reported value depends on the sample handling and its denominator. Saraci and colleagues compared single-platform flow cytometry with next-generation flow (NGF) in a prospective clinical-trial setting involving transplant-ineligible patients with newly diagnosed multiple myeloma. Their study is published in Cytometry Part B: Clinical Cytometry (2026), DOI 10.1002/cyto.b.70068. A result called a CTC percentage is not automatically portable between the two methods.

The study design: distinguish the full cohort from the paired comparison

The REAL MM study provided a 168-patient cohort for the broader analysis, but the head-to-head comparison between methods used blood from only 34 patients assessed at screening. That paired subgroup is the appropriate denominator for direct method comparison. Extending findings from 34 paired samples to every clinical state, treatment phase or myeloma population would overstate the evidence.

The single-platform approach used counting beads to estimate absolute CTC concentration in peripheral blood, together with a separate clonality assessment. The NGF approach incorporated bulk cell processing and a higher number of acquired nucleated cells, including fixation and permeabilisation steps not needed for the single-platform count. These workflows differ in more than fluorochrome count. They can also differ in sample recovery, processing losses and what is placed in the final denominator.

Paired detection was higher with NGF

In the 34 paired patients, single-platform flow detected circulating tumour plasma cells in 29/34 (85.3%), compared with 31/34 (91.2%) using NGF. The two additional NGF-positive samples were present at very low tumour-cell abundance. NGF acquired a median of approximately 10.5 million nucleated cells in the paired analysis, versus approximately 0.94 million using single-platform flow. The study reported a median limit of detection of about 0.0002% for NGF versus 0.0021% for conventional flow; median limits of quantification were about 0.0005% and 0.0053%, respectively. These method-specific estimates are not universal laboratory performance claims.

A lower limit of detection supports more reliable detection of very rare cells. It does not imply that the more sensitive workflow will necessarily report a larger percentage when the same patient is tested.

The enumeration paradox: high correlation is not agreement

Despite higher analytical sensitivity, NGF reported a lower median CTC proportion than single-platform flow in the paired subgroup: approximately 0.0049% versus 0.009%. The reported median ratio of NGF to single-platform percentages was around 0.51. Median absolute counts were approximately 0.3 versus 0.55 cells per microlitre. The methods showed a high correlation, but correlation alone does not establish agreement or justify substituting one measurement for another.

A plausible source of systematic differences is sample processing: the NGF workflow requires more extensive handling, and fixation/permeabilisation can change cell recovery. Those explanations should be treated as methodological considerations, not as proof that a particular processing step caused the entire observed difference. Laboratories should examine paired bias, counting and gating denominators, losses in rare-cell recovery, and clinically relevant classification disagreements before considering two assays interchangeable.

What should be recorded before comparing CTC results

For each method document the blood volume, anticoagulant, preanalytical delay, viability, lysis and wash steps, presence of fixation/permeabilisation, reference count or beads, number of nucleated events analysed, antigen-based gating strategy, clonality confirmation and the reported unit. State whether the result is a percentage of analysed nucleated cells, an absolute concentration per microlitre or a qualitative detected/not-detected outcome. Do not silently convert between these measurands.

When evaluating an internal method comparison, choose suitable low-concentration samples, report paired differences as well as correlations, and establish a laboratory-specific acceptability criterion. A two-fold proportional difference near a clinical threshold may matter even where correlation is excellent. Also distinguish the analytical limit of detection from a prognostic cut-off or a clinical decision threshold.

Clinical limits: what this article does not establish

The comparison concerned patients at diagnosis, not a prospective validation of post-treatment peripheral-blood measurable residual disease. The study does not establish that a baseline CTC percentage from either platform can replace marrow MRD analysis, nor does it validate a universal clinical threshold transferable across laboratories. Biological interpretation and prognosis depend on population, treatment, follow-up, method and timing.

Daily Learning Pill: sensitivity and recovery answer different questions

Ask two questions separately. How few abnormal cells can the assay detect with adequate confidence? And how many of the cells initially present survive processing and enter the final calculation? A workflow may detect rarer events yet enumerate a lower percentage because its specimen handling and denominator differ. Neither result is necessarily wrong; they are not automatically the same measurand.

Practical next step

Before adopting a published peripheral-blood CTC threshold, verify the exact method on your own samples, including rare-cell precision, recovery, denominator definition and paired-bias behaviour. Where local evidence is absent, report method-specific values and their limitations rather than claiming interchangeability. This CFCM Technical Review is an original educational interpretation of a published study, not an independent peer review, external clinical validation or a recommendation to change patient management. Related clinical flow cytometry resources are available at cfcm.es.