“Near-Infrared Fluorescence Lymphatic Imaging to Reconsider Occlusion Pressure of Superficial Lymphatic Collectors in Upper Extremities of Healthy Volunteers”: Facts and/or Artifact?

As specialists in imaging, especially of the lymphatic system, we read with some irritation the article by Belgrado et al. ¹

1. When analyzing any study using one imaging approach, we have to be able to find the responses to the following question: “How is what is reported technically seen?”

Back to basics: “With NIR imaging after (intradermal) injection of ICG, one camera sees (obtains one indirect image of) photons emitted by molecules of ICG excited by photons emitted by one laser.”

Because the number of photons emitted by (a given number of) indocyanine green (ICG) molecules in one precise volume depends on the number of “exciting” photons illuminating these molecules, the “intensity” of the laser (of their Photodynamic Eye [PDE] system) should have been mentioned by the authors (this problem is only raised by the authors when they write “the distal lymphatic seemed widened, but as we reduced infrared light intensity, the vessels' walls reappeared as initially,” which supports our hypothesis raised in the next point and what we observed with our experimental protocol in Supplementary Data; Supplementary Data are available in the online article at www.liebertpub.com/lrb).

But the “final” image of the “object” rendered by the camera system (their “video”) will also depend on the “sensitivity” and “contrast” used to display the images, parameters also not mentioned in their article (as standardized either from one patient to another or in the same patient over time?).

2. What do they see?

The authors (report to) see (rhythmic) variations (and changes) of fluorescence in one considered lymphangion.

We would agree with the authors that these rhythmic changes in fluorescence correspond to ICG liquid “lymph bolus passages” but under 20–30 mm Hg.

However, with under 40 mm Hg and more, these changes do not seem to imply “fluorescent lymph … passages,” more exactly, the inflows (and the passages) of “bolus-volume” of new ICG fluorescent lymph in the considered lymphangion.

What is observed might simply correspond to the contraction of the lymphatic vessel (of the vascular walls coated by ICG) (the author reported to us and we personally observed such a phenomenon on near-infrared [NIR] imagings obtained with ICG days after its injection).

The authors indeed imaged lymphatic vessels (LV) in which ICG has already transited and, when they begin to apply increasing pressures on these LV, these LV are (already) fluorescent (it is a pity that the authors do not report any data about the fluorescence seen in the LV before the application of the pressures and, most importantly, about the amount of ICG in the noncompressed LV).

When the lymphangion is contracting under the occlusive pressure (without propelling any fluorescent lymph and/or liquid), it corresponds to one change in the apparent diameter of the vessel (and in one change in the volume in which the number of ICG molecules coating the vascular walls remains constant)… and this change is enough to give the variation in the fluorescence observed.

Our hypothesis was verified by performing (see Supplementary Data for the details and for some images) the following experience:

• We put in two flexible catheters (5 cm length) of different diameters (25 and 11 mm) the same amount of ICG (25 ng in 0.05 and 0.25 mL) resulting in fact in one higher concentration of ICG per cm (in the finest catheter) mimicking the decrease of volume of the contracting vessel under pressure without any entry of new ICG and only the three-dimensional changes of the ICG coated on the vascular surfaces.

• We used the same (PDE) camera as Belgrado's team and imaged the two fluorescent tubes simply with variations of the intensity of the laser.

And we obtained … dramatic changes in what we saw imaged when the laser intensity varied from 1 to 10 … with the “large” vessel being not visible and the thin “vessel” remaining visible (see figure and table in Supplementary Data).

So and in other words, it means that according to the intensity of the laser (some amounts of), ICG coating the vascular lymphatic walls will not be visible when the vessel is at a given diameter and will be visible when the diameter is diminished by a factor 2 … and that consequently you will have the impression of one fluorescence progression, corresponding in fact to the propagation of one lymphatic contraction, but not to the progression of one new lymph bolus in the vessel.

So, we would agree that Belgrado's team saw (at least) the contractions of the LV under pressures up to 80–90 mm Hg, but what they report to see is no proof that these contractions (and their maneuvers) have propelled (ICG fluorescent) lymph (liquids and/or proteins) under their cuff at these pressures … what is finally the most important.

To conclude, their work presents inherent technical limitations and any conclusion based on their results seems to us ill-founded. So far, the realization of future and/or of ongoing works with the same technique by Belgrado's team might also be questioned.

Additional Remarks, Questions, and Comments

(a) The LV “seem” to be no longer able to contract under more than 80–90 mmHg. Such “high” pressures might be not damaging for normal LV in these healthy subjects and when applied during only several minutes, but what about with such high pressures repeated (sometimes for hours) on diseased lymphatics-limbs (as encountered in clinical situations)?

(b) “Lymph bolus passage”… “appeared approximately every 30 sec under baseline conditions”: the normal rhythm of lymphatic contractions is, for instance with hand-grip, higher than the 2/min under the authors' “fill and flush.” Does their 2/min frequency correspond to the rhythm of their “fill and flush” and/or do they conclude that their “maneuvers” do worse than natural contractions in terms of frequency in these normal subjects?

(c) In their discussion about ICG toxicity on lymphatics, the authors failed to mention an article that highlighted problems in ICG imaging, published in this Journal. ²

(d) “No significant difference was found between age group, gender, or limb side”: the numbers of volunteers in the groups are small and, again, these conclusions seem ill-founded to us.

(e) “… the vessels' walls reappeared …”: the resolution of the PDE system does not allow discernment of the lymphatic walls.