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344
flexibility. The main limitation of this method is
lack of flexibility of bonded tissue [36].
Dye-enhanced tissue welding: The concept of
using a topical tissue-staining dye to facilitate
selective delivery of laser energy by the target tis-
sue has been postulated to improve tissue weld-
ing. A nontoxic dye that is strongly absorbed by a
specific laser wavelength can serve to confine
photon absorption and the resultant thermal
energy to the weld site. A variety of combinations
of dyes and lasers have been studied with vari-
able success rates [17, 33, 37, 38]. It seems that
combination of indocyanine green with either
pulsed alexandrite or pulsed diode laser is supe-
rior to other dye-enhanced tissue welding tech-
niques. Nonetheless, it is worth noting that very
limited clinical data have been available yet that
confirm the clinical value of dye-enhanced tissue
welding.
Nanoshells are a new class of nanoparticles
consisting of a dielectric core surrounded by a
thin metal shell. Use of gold nanoshells in con-
junction with near Infrared light has recently
been suggested as a means of dye-enhanced tis-
sue welding. Application of lasers at wavelengths
within the near infrared, the region between
approximately 650 and 900  nm, where tissue
components have minimal absorption, decreases
the chance of widespread thermal damage and
improves penetration depth [39]. The use of
nanoshells has several advantages over indocya-
nine green. For example, the small size of
nanoshells reduces diffusion from the site of
treatment and concentrates heating at the inter-
face to be welded. Also they are less photosensi-
tive hydrolytically sensitive and susceptible to
photobleaching in the presence of light compar-
ing to indocyanine green.
Addition of Growth factors: Attempts have
been made to use recombinant growth factors, as
an adjunct to laser-assisted tissue soldering to
accelerate wound healing. A variety of growth
factors such as HB-EGF, FGF, TGF-β, etc. have
been studied. The result of an animal study by
Poppas and colleagues showed that addition of
TGF-β to the solder (albumin in their study)
increases the tensile strength of the wound by
more than 50%. Using this technique, it is imper-
ative to maintain a predetermined tissue tempera-
ture in order to prevent thermal degradation of
growth factors [40].
Lasers vs. Conventional Methods
of Acute Wound Closure
Conventional techniques for tissue bonding
(sutures, staples, and adhesives) are highly reliable
procedures that have proven themselves over the
years to be good clinical practice. Sutures have
been successfully used for centuries. They are
inexpensive, flexible, reliable, and readily avail-
able [15]. However, they are not the perfect tech-
nique due to several reasons (Table 19.2). Since
sutures cause trauma to the skin, and ­introduce
foreign body, they can result in inflammation,
granuloma formation, and scarring. Many techni-
cal factors such as position on the needle in the
holder, the slope of the tissue at needle entrance,
suture spacing, knot tension, and choice of suture
material can affect wound healing [25, 41]. Staples
are another mean of wound closure which share
many common characteristics with sutures.
However, they are faster and more uniform than
sutures. Their main disadvantage is that they come
in predetermined size which precludes their use in
some anatomical sites. Adhesives are clean, fast,
non-operator-dependant, painless method of
wound closure. They are an excellent “no needle”
alternative in pediatric patients. However, for most
applications, they have not been able to provide
adequate strength [15, 42, 43].
As shown in detail in this chapter, laser-
assisted tissue bonding can transcend the limita-
tions of conventional methods in many aspects.
Their potential advantages over conventional
methods include increased immediate wound
strength, fluid-tight closure, decreased operative
repair time, reduced probability of infection and
bleeding,
and
improved
cosmetic
results.
However, there have been several obstacles
which prevented physicians from using laser
welding clinically. These included collateral ther-
mal injuries, inconsistency of the results, and a
lack of understanding of the exact mechanism by
which laser irradiation induces tissue bonding. In
E. Azimi et al.

345
addition, there are many parameters that need to
be optimized in the welding process. These
parameters include wavelength, fluence, pulse
duration, repetition rate, irradiation time, spot
size, and solder selection. Indeed, the parameter
window for optimum tissue bonding is very
small. All parameters should be chosen appropri-
ately to provide enough heat for denaturation and
crosslinking of collagen fibers, but not to the
level of tissue necrosis and sloughing of wound
edges. What makes the use of laser even more
complicated is the fact that energy levels and
exposure times that may work very well with cer-
tain tissues may not be the best for other situa-
tions [15, 19]. As we discuss later in this chapter,
several thermal feedback systems have been sug-
gested to overcome the above limitations.
Mechanism of Laser-Assisted Wound
Bonding
The exact mechanism involved in laser-assisted
wound closure is not completely understood.
Nonetheless, what is commonly believed is that
tissue bonding occurs mainly due to the thermal
effect of laser. The heat produced by laser energy
in the tissue causes collagen fibers to loose their
triple helix structure and become fused, inter-
twined, swollen, and dissolved. This generates a
coagulum that serves both as a coating for sealing
the wound and as a sophisticated scaffold for re-
colonization of cells, as in the case of re-epitheli-
alization [26, 44, 45]. Other theories have been
postulated, as to say Helmsworth and colleagues
believe that welding effect is the result of reorga-
Table 19.2  Advantages and disadvantages of different methods of wound closure
Advantage
Disadvantage
Suture
Reliable
Cause trauma to the tissue
Flexible
Time consuming
Inexpensive
Operator dependant
Available
Introduce foreign body
No immediate watertight closure
Risk of needle-stick
Risk of infection (due to lack of sealing)
Need suture removal
Staple
Reliable
Cause trauma to the tissue
Available
Inflexible (predetermined size)
Relatively quick
Introduce foreign body
Not operator dependant
No immediate watertight closure
Risk of needle-stick
Risk of infection (due to lack of sealing)
Need staple removal
Adhesive
Immediate watertight closure
Expensive (controversial)
Painless
Does not provide hemostasis
No trauma to tissue
Introduce foreign body
“No needle” procedure
(Possible) need for subcutaneous sutures
Less risk of infection
Risk of tissue reactivity
No need for removal
Not flexible (comparing to sutures)
Fast
Laser
Immediate watertight closure
Expensive
Better scar
Not readily available
“No needle” procedure
Foreign body (Soldering, dye-enhanced)
Less risk of infection
Risk of dehiscence
No need for removal
Complicated (many parameters to consider)
Fast
Risk of thermal damage
Dynamic effects (may increase growth
factors)
Inconsistent results
19  Laser and Light for Wound Healing Stimulation

346
nization of intramolecular disulfide bonds of
laminin, type IV collagen, and entactin rather
than covalent bonds of type I collagen [46].
Despite the controversies, it seems that collagen
plays a major role although bonding is most
likely dependant on extracellular proteins rather
than collagen alone.
Laser Systems and Parameters
for Optimal Wound Bonding
In order to achieve a reliable tensile strength, it
is crucial to estimate the optimal photonic
energy that is to be delivered to tissue. In this
respect, major determining factors are laser
wavelength, power, exposure time, and mode of
operations (continuous wave or pulsed). For
this reason, various types of laser systems were
investigated [15, 25]. CO2 was one of the first
lasers employed for wound bonding because of
its availability. However, it is a poor choice of
wavelength and unlikely to yield a reliable high
strength bonds. Due to the high absorption at
the surface, the outermost tissue layers are
“overcooked,” whereas the deeper layers are
hardly affected at all. Therefore, the tempera-
ture in dermis is not high enough for collagen
alteration and fusion. If the energy is increased
to achieve the suitable temperature in dermis,
the surface will burn. Increasing the pulse dura-
tion will also result in producing a large zone of
thermal damage. Like CO2 laser, holmium:YAG
(Ho:YAG) has a high absorption by water,
hence causes thermal injury at the surface. To
overcome this problem, these lasers need to be
used under a temperature-controlled system.
Near Infrared lasers (650–900  nm) such as
diode lasers are also becoming more popular in
welding
studies. While
the
wavelengths
between 780 and 850 nm have the advantage of
less absorption by water, their relatively high
absorption by melanin prevents their deeper
penetration [47, 48]. Recently, 980-nm diode
was suggested as a better wavelength for wound
closure due to its better absorption by water and
less absorption by melanin comparing to other
infrared lasers [48]. In general, although near
infrared lasers would allow deeper penetration
of the light, their absorption by tissue compo-
nents is minimal; hence, they may need to be
used in conjunction with exogenous absorbers
to induce welding. Due to its water and melanin
absorption coefficient values [47], Nd:YAG
(1064 nm) is another infrared laser used in tis-
sue welding. Like many of the near infrared
lasers, Nd:YAG laser needs to be used with sol-
ders for optimal welding. It should be noted
that most of our knowledge about lasers used
for wound bonding is on either in-vitro or ani-
mal studies. Therefore, the optimal laser sys-
tems and parameters for human wound closure
yet to be determined.
Lasers for Chronic Wounds
Laser irradiation was introduced as a noninva-
sive therapeutic modality for acceleration of
wound healing in the late 1960s, and has since
been used for the treatment of a variety of
chronic ulcers [49]. Different laser systems such
as helium-neon, gallium-arsenide (GaAs), gal-
lium-aluminum-arsenide (GaAlAs), Nd:YAG,
carbon dioxide, ruby, krypton, and argon dye
lasers have been studied [50, 51]. Despite differ-
ences in wavelengths, the common characteristic
of all these lasers is that they all employ low
energy for wound healing. This method of ther-
apy has been known as low-intensity, low-power,
or low-level laser therapy. It has been suggested
to increase the speed, quality and tensile strength
of tissue repair, resolve inflammation and pro-
vide pain relief. The use of low intensity laser
therapy is not limited to wound healing; and it
has been used in odontological, rehabilitative,
and other medical specialties. The basic princi-
ple of laser therapy is that low intensity laser
radiation has the capability to alter cellular
behavior in the absence of significant heating.
Previous works have been focused on three
areas: in-vitro studies on molecular and cellular
function, animal studies, and human trials.
Unfortunately, the clinical data is mostly anec-
dotal, poorly controlled, and more variable than
might be desired. The in-vitro and animal studies
are less arguable, and provide most of the scien-
tific rationale of laser therapy.
E. Azimi et al.

347
Lasers have been used for healing of a variety
of chronic wounds such as pressure ulcers,
venous ulcers, and diabetic ulcers [Table 19.3].
The first implication of lasers for pressure ulcers
was to use them as a surgical tool for debride-
ment. Controlled trials on CO2 laser versus con-
ventional debridement showed less bleeding, less
infection, and shorter hospital stay with the use
of CO2 laser [52, 53]. Later, low intensity lasers
such as diode and GaAs were attempted. While
some reports found impressive wound healing
outcomes, some others showed no advantages
[54–56]. There is insufficient evidence to suggest
a benefit of treating venous ulcers with low-
intensity laser therapy. Most of the data are anec-
dotal, and there is only one small randomized
controlled trial suggesting the therapeutic benefit
of laser therapy for venous ulcers [57]. Low
intensity laser therapy has been shown by various
studies to be effective in the treatment of diabetic
wound healing [58]. Some of the suggested
advantages of this method are increased micro-
circulation, increased speed of healing, improved
wound epithelialization, increased granulation
tissue formation, and increased collagen deposi-
tion [59–61]. However, some authors emphasize
that depending on the applied dose, wavelength,
irradiation time, and also the conditions of the
treated tissue, different positive and negative bio-
logical answers can be achieved.
Mechanism of Laser-Assisted
Wound Healing
The exact mechanism of action of low intensity
laser therapy is not completely understood.
Currently there is no accepted theory to explain the
mechanism of low-intensity lasers, and this lack of
knowledge complicates the evaluation of conflict-
ing reports in the literature. Another limitation is
the lack of ideal models of chronic wounds. Most
of the studies have been conducted on surgically
excised skin. These wound models excluded com-
mon problems associated with delayed healing,
such as ischemia, infection, and necrotic debris.
What makes the data even more confusing is that a
variety of laser parameters such as wavelength, flu-
ence, and time of treatment onset can influence the
biologic effects of low intensity lasers.
The suggested mechanisms at molecular level
are stimulation of Ca influx and mitosis rate,
increased expression of Heat-Shock-Proteins
(HSP70 is overexpressed in the laser-induced
thermal damage zone and HSP47 is expressed
during the recovery phase [62]), increased expres-
sion of growth factors such as TGF-β, alteration
of mitochondrial activity and increased ATP syn-
thesis, augmented formation of mRNA and pro-
tein secretion. On the cellular level laser-induced
changes are enhancement of fibroblast and kerati-
nocyte proliferation and migration, angiogenesis,
improvement of phagocytosis, and increased rate
of transformation of fibroblasts into myofibro-
blasts (Fig.  19.2) [62–70]. Some of the animal
studies on laser-assisted wound healing are in
accordance of molecular and cellular studies,
showing
decreased
inflammatory
period,
increased collagen and granulation tissue in the
wound bed, increased tensile strength, and faster
epithelialization [55, 71, 72]. However, repeated
experiments on many of the above in-vitro and
animal models have failed to verify these benefits.
These conflicting reports may partly be explained
by considering discordance among the laser types
Table 19.3  Commonly used lasers for different types of
chronic ulcers
Type of
chronic
ulcer
Laser type and
wavelength
Energy (J/
cm2)
Resulta
Venous
810 nm
GaA1As
4.0
Not effective
660–950 nm
GaA1As
12.0
Effective
904 nm GaAs
1.0
Effective
632.8 nm
HeNe
4.0
Effective
810 nm diode
4.0
Effective
Pressure
904 nm GaAs
1.0
Effective
830 nm diode
5.0
Effective
Diabetic
670 nm diode
18 and 36
Not effective
with 36 J/cm2
632.8 nm
HeNe
1.0, 4.0,
4.8, 5.0,
10.0, and
16.0
Effective with
all except
16 J/cm2
904 nm GaAs
1.0
Effective
830 nm diode
5.0
Effective
aThe results given are based on authors’ conclusion. Not
all the studies are well controlled randomized trials
19  Laser and Light for Wound Healing Stimulation

348
used, the parameters selected, and the wound
models chosen. In summary, to better understand
the role of low intensity lasers in healing of
chronic wounds, well-controlled studies that cor-
relate cellular effects [55] and biologic processes
are needed. In the absence of such studies, the lit-
erature does not appear to support widespread use
of lasers in wound healing at this time.
References
1. Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert
L, Hunt TK, et al. Human skin wounds: a major and
snowballing threat to public health and the economy.
Wound Repair Regen. 2009;17(6):763–71.
2. Harding KG, Morris HL, Patel GK.  Science, medi-
cine and the future: healing chronic wounds. BMJ.
2002;324(7330):160–3.
3. Frykberg RG, Zgonis T, Armstrong DG, Driver VR,
Giurini JM, Kravitz SR, et al. Diabetic foot disorders.
A clinical practice guideline (2006 revision). J Foot
Ankle Surg. 2006;45(5 Suppl):S1–66.
4. de Araujo T, Valencia I, Federman DG, Kirsner
RS.  Managing the patient with venous ulcers. Ann
Intern Med. 2003;138(4):326–34.
5. Bolognia J, Jorizzo JL, Rapini RP.  Dermatology.
London: Mosby; 2003.
6. Clark RA, Ghosh K, Tonnesen MG.  Tissue engi-
neering for cutaneous wounds. J Invest Dermatol.
2007;127(5):1018–29.
7. Werner S, Krieg T, Smola H. Keratinocyte-fibroblast
interactions in wound healing. J Invest Dermatol.
2007;127(5):998–1008.
8. Mast BA, Schultz GS.  Interactions of cytokines,
growth factors, and proteases in acute and chronic
wounds. Wound Repair Regen. 1996;4(4):411–20.
9. Booth BA, Polak KL, Uitto J. Collagen biosynthesis by
human skin fibroblasts. I. Optimization of the culture
conditions for synthesis of type I and type III procol-
lagens. Biochim Biophys Acta. 1980;607(1):145–60.
10. Lazarus GS, Cooper DM, Knighton DR, Margolis DJ,
Pecoraro RE, Rodeheaver G, et  al. Definitions and
guidelines for assessment of wounds and evaluation
of healing. Arch Dermatol. 1994;130(4):489–93.
Tissue injury
Blood extravasation
Inflammatory phase
Laser
Platelet
Neutrophil
Macrophage
Epithelial cell
Fibroblast
Proliferative phase
Remodeling phase
Day 14
Day 7
Day 1
Minutes to hours
0
Months
Phagocytose bacteria
and matrix proteins
Angiogenesis
PDGF
EGF
TGF-β
IGF-1
Serotonin
Fibrinogen
ADP
TXA2
PDGF
FGF
TGF-β
TGF-α
VEGF
Migration and proliferation
IL-1
IL-6
PDGF
GM-CSF
Collagenase,
hyaluronidase
Collagen III degradation
Collagen remodeling
Collagen I synthesis
KGF
HB-EGF
Activin
TGF-β
Migration and proliferation
TGF-α
Fig. 19.2  The molecular and cellular effects of low
intensity lasers on chronic wounds: Lasers mainly affect
inflammatory and proliferative phases of wound healing.
At cellular level, lasers improve phagocytosis, enhance
angiogenesis, and increase proliferation of fibroblasts and
keratinocytes. At molecular level, they increase FGF,
TGF-β, VEGF, IL-6, and PDGF. IL interleukin, FGF
fibroblast growth factor, VEGF vascular endothelial
growth factor, PDGF platelet-derived growth factor, TGF-
β transforming growth factor-beta
E. Azimi et al.

349
11. Steed DL, Hill DP, Woodske ME, Payne WG, Robson
MC.  Wound-healing trajectories as outcome mea-
sures of venous stasis ulcer treatment. Int Wound J.
2006;3(1):40–7.
12. Williams JZ, Barbul A. Nutrition and wound healing.
Surg Clin North Am. 2003;83(3):571–96.
13. Crowe MJ, Doetschman T, Greenhalgh DG. Delayed
wound healing in immunodeficient TGF-beta 1
knockout mice. J Invest Dermatol. 2000;115(1):3–11.
14. Mester E, Spiry T, Szende B, Tota JG. Effect of laser
rays on wound healing. Am J Surg. 1971;122(4):532–5.
15. Bass LS, Treat MR. Laser tissue welding: a compre-
hensive review of current and future clinical applica-
tions. Lasers Surg Med. 1995;17(4):315–49.
16. Fried NM, Walsh JT Jr. Laser skin welding: in vivo
tensile strength and wound healing results. Lasers
Surg Med. 2000;27(1):55–65.
17. Kirsch AJ, Cooper CS, Gatti J, Scherz HC, Canning
DA, Zderic SA, et  al. Laser tissue soldering for
­hypospadias repair: results of a controlled prospective
clinical trial. J Urol. 2001;165(2):574–7.
18. Kaviani A, Djavid GE, Ataie-Fashtami L, Fateh M,
Ghodsi M, Salami M, et  al. A randomized clinical
trial on the effect of low-level laser therapy on chronic
diabetic foot wound healing: a preliminary report.
Photomed Laser Surg. 2011;29(2):109–14.
19. Simhon D, Halpern M, Brosh T, Vasilyev T, Ravid A,
Tennenbaum T, et al. Immediate tight sealing of skin
incisions using an innovative temperature-controlled
laser soldering device: in vivo study in porcine skin.
Ann Surg. 2007;245(2):206–13.
20. Sigel B, Acevedo FJ. Vein anastomosis by electroco-
aptive union. Surg Forum. 1962;13:233–5.
21. Garden JM, Robinson JK, Taute PM, Lautenschlager
EP, Leibovich SJ, Hartz RS. The low-output carbon
dioxide laser for cutaneous wound closure of scal-
pel incisions: comparative tensile strength studies of
the laser to the suture and staple for wound closure.
Lasers Surg Med. 1986;6(1):67–71.
22. Abergel RP, Lyons RF, White RA, Lask G, Matsuoka
LY, Dwyer RM, et al. Skin closure by Nd:YAG laser
welding. J Am Acad Dermatol. 1986;14(5 Pt 1):810–4.
23. Wang S, Grubbs PE Jr, Basu S, Robertazzi RR,
Thomsen S, Rose DM, et al. Effect of blood bonding
on bursting strength of laser-assisted microvascular
anastomoses. Microsurgery. 1988;9(1):10–3.
24. McKennan
KX.
“Tissue
welding”
with
the
argon laser in middle ear surgery. Laryngoscope.
1990;100(11):1143–5.
25. Talmor M, Bleustein CB, Poppas DP.  Laser tissue
welding: a biotechnological advance for the future.
Arch Facial Plast Surg. 2001;3(3):207–13.
26. Tang J, Godlewski G, Rouy S, Delacretaz
G.  Morphologic changes in collagen fibers after
830  nm diode laser welding. Lasers Surg Med.
1997;21(5):438–43.
27. Jain KK, Gorisch W. Repair of small blood vessels
with the neodymium-YAG laser: a preliminary report.
Surgery. 1979;85(6):684–8.
28. Capon A, Mordon S. Can thermal lasers promote skin
wound healing? Am J Clin Dermatol. 2003;4(1):1–12.
29. Abergel RP, Lyons R, Dwyer R, White RR, Uitto
J.  Use of lasers for closure of cutaneous wounds:
experience with Nd:YAG, argon and CO2 lasers. J
Dermatol Surg Oncol. 1986;12(11):1181–5.
30. Massicotte JM, Stewart RB, Poppas DP.  Effects of
endogenous absorption in human albumin solder
for acute laser wound closure. Lasers Surg Med.
1998;23(1):18–24.
31. Lauto A. Repair strength dependence on solder pro-
tein concentration: a study in laser tissue-welding.
Lasers Surg Med. 1998;22(2):120–5.
32. Levanon D, Katzir A, Ravid A.  A scanning elec-
tron microscopy study of CO2 laser-albumin sol-
dering in the rabbit model. Photomed Laser Surg.
2004;22(6):461–9.
33. Wider TM, Libutti SK, Greenwald DP, Oz MC, Yager
JS, Treat MR, et al. Skin closure with dye-enhanced
laser welding and fibrinogen. Plast Reconstr Surg.
1991;88(6):1018–25.
34. Lauto A, Foster LJ, Ferris L, Avolio A, Zwaneveld N,
Poole-Warren LA. Albumin-genipin solder for laser
tissue repair. Lasers Surg Med. 2004;35(2):140–5.
35. Birch JF, Mandley DJ, Williams SL, Worrall DR,
Trotter PJ, Wilkinson F, et  al. Methylene blue
based protein solder for vascular anastomoses: an
in  vitro burst pressure study. Lasers Surg Med.
2000;26(3):323–9.
36. Lauto A, Kerman I, Ohebshalon M, Felsen D, Poppas
DP. Two-layer film as a laser soldering biomaterial.
Lasers Surg Med. 1999;25(3):250–6.
37. DeCoste SD, Farinelli W, Flotte T, Anderson RR. Dye-
enhanced laser welding for skin closure. Lasers Surg
Med. 1992;12(1):25–32.
38. Small W IV, Heredia NJ, Maitland DJ, Da Silva LB,
Matthews DL.  Dye-enhanced protein solders and
patches in laser-assisted tissue welding. J Clin Laser
Med Surg. 1997;15(5):205–8.
39. Gobin AM, O’Neal DP, Watkins DM, Halas NJ,
Drezek RA, West JL. Near infrared laser-tissue weld-
ing using nanoshells as an exogenous absorber. Lasers
Surg Med. 2005;37(2):123–9.
40. Poppas DP, Massicotte JM, Stewart RB, Roberts AB,
Atala A, Retik AB, et al. Human albumin solder sup-
plemented with TGF-beta 1 accelerates healing fol-
lowing laser welded wound closure. Lasers Surg Med.
1996;19(3):360–8.
41. Seki S.  Techniques for better suturing. Br J Surg.
1988;75(12):1181–4.
42. Gennari R, Rotmensz N, Ballardini B, Scevola S,
Perego E, Zanini V, et al. A prospective, randomized,
controlled clinical trial of tissue adhesive (2-octylcya-
noacrylate) versus standard wound closure in breast
surgery. Surgery. 2004;136(3):593–9.
43. Ong CC, Jacobsen AS, Joseph VT. Comparing wound
closure using tissue glue versus subcuticular suture
for pediatric surgical incisions: a prospective, ran-
domised trial. Pediatr Surg Int. 2002;18(5–6):553–5.
44. Schober R, Ulrich F, Sander T, Durselen H, Hessel
S.  Laser-induced alteration of collagen substruc-
ture allows microsurgical tissue welding. Science.
1986;232(4756):1421–2.
19  Laser and Light for Wound Healing Stimulation

350
45. Simhon D, Brosh T, Halpern M, Ravid A, Vasilyev T,
Kariv N, et al. Closure of skin incisions in rabbits by
laser soldering: I: Wound healing pattern. Lasers Surg
Med. 2004;35(1):1–11.
46. Helmsworth TF, Wright CB, Scheffter SM, Schlemm
DJ, Keller SJ.  Molecular surgery of the basement
membrane by the argon laser. Lasers Surg Med.
1990;10(6):576–83.
47. Peavy GM.  Lasers and laser-tissue interaction. Vet
Clin North Am Small Anim Pract. 2002;32(3):517–
34, v–vi.
48. Gulsoy M, Dereli Z, Tabakoglu HO, Bozkulak
O. Closure of skin incisions by 980-nm diode laser
welding. Lasers Med Sci. 2006;21(1):5–10.
49. Mester E, Mester AF, Mester A.  The biomedi-
cal effects of laser application. Lasers Surg Med.
1985;5(1):31–9.
50. Wheeland RG.  Lasers for the stimulation or inhi-
bition of wound healing. J Dermatol Surg Oncol.
1993;19(8):747–52.
51. Kawalec JS, Hetherington VJ, Pfennigwerth TC,
Dockery DS, Dolce M.  Effect of a diode laser on
wound healing by using diabetic and nondiabetic
mice. J Foot Ankle Surg. 2004;43(4):214–20.
52. Dixon JA. Current laser applications in general sur-
gery. Ann Surg. 1988;207(4):355–72.
53. Juri H, Palma JA. CO2 laser in decubitus ulcers: a com-
parative study. Lasers Surg Med. 1987;7(4):296–9.
54. Schindl A, Schindl M, Schindl L.  Successful treat-
ment of a persistent radiation ulcer by low power laser
therapy. J Am Acad Dermatol. 1997;37(4):646–8.
55. Allendorf JD, Bessler M, Huang J, Kayton ML, Laird
D, Nowygrod R, et al. Helium-neon laser irradiation
at fluences of 1, 2, and 4 J/cm2 failed to acceler-
ate wound healing as assessed by both wound con-
tracture rate and tensile strength. Lasers Surg Med.
1997;20(3):340–5.
56. Basford JR.  Low-energy laser therapy: controver-
sies and new research findings. Lasers Surg Med.
1989;9(1):1–5.
57. Flemming KA, Cullum NA, Nelson EA. A systematic
review of laser therapy for venous leg ulcers. J Wound
Care. 1999;8(3):111–4.
58. Beckmann KH, Meyer-Hamme G, Schroder S. Low
level laser therapy for the treatment of diabetic foot
ulcers: a critical survey. Evid Based Complement
Alternat Med. 2014;2014:626127.
59. Schindl A, Heinze G, Schindl M, Pernerstorfer-
Schon H, Schindl L. Systemic effects of low-intensity
laser irradiation on skin microcirculation in patients
with diabetic microangiopathy. Microvasc Res.
2002;64(2):240–6.
60. Reddy GK, Stehno-Bittel L, Enwemeka CS.  Laser
photostimulation accelerates wound healing in dia-
betic rats. Wound Repair Regen. 2001;9(3):248–55.
61. Yu W, Naim JO, Lanzafame RJ.  Effects of photo-
stimulation on wound healing in diabetic mice. Lasers
Surg Med. 1997;20(1):56–63.
62. Sajjadi AY, Mitra K, Grace M.  Expression of heat
shock proteins 70 and 47 in tissues following short-
pulse laser irradiation: assessment of thermal damage
and healing. Med Eng Phys. 2013;35(10):1406–14.
63. Abergel RP, Meeker CA, Lam TS, Dwyer RM,
Lesavoy MA, Uitto J.  Control of connective tissue
metabolism by lasers: recent developments and future
prospects. J Am Acad Dermatol. 1984;11(6):1142–50.
64. Pourreau-Schneider N, Ahmed A, Soudry M,
Jacquemier J, Kopp F, Franquin JC, et  al. Helium-
neon laser treatment transforms fibroblasts into myo-
fibroblasts. Am J Pathol. 1990;137(1):171–8.
65. Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt
S, Alam M.  Low-level laser therapy for wound
healing: mechanism and efficacy. Dermatol Surg.
2005;31(3):334–40.
66. Pereira AN, Eduardo Cde P, Matson E, Marques
MM.  Effect of low-power laser irradiation on cell
growth and procollagen synthesis of cultured fibro-
blasts. Lasers Surg Med. 2002;31(4):263–7.
67. Medrado AR, Pugliese LS, Reis SR, Andrade
ZA.  Influence of low level laser therapy on wound
healing and its biological action upon myofibroblasts.
Lasers Surg Med. 2003;32(3):239–44.
68. Schindl A, Merwald H, Schindl L, Kaun C, Wojta
J. Direct stimulatory effect of low-intensity 670 nm
laser irradiation on human endothelial cell prolifera-
tion. Br J Dermatol. 2003;148(2):334–6.
69. Ogita M, Tsuchida S, Aoki A, Satoh M, Kado S,
Sawabe M, et  al. Increased cell proliferation and
differential protein expression induced by low-
level Er:YAG laser irradiation in human gingival
fibroblasts: proteomic analysis. Lasers Med Sci.
2015;30(7):1855–66.
70. Dungel P, Hartinger J, Chaudary S, Slezak P, Hofmann
A, Hausner T, et al. Low level light therapy by LED
of different wavelength induces angiogenesis and
improves ischemic wound healing. Lasers Surg Med.
2014;46(10):773–80.
71. Lyons RF, Abergel RP, White RA, Dwyer RM, Castel
JC, Uitto J. Biostimulation of wound healing in vivo by
a helium-neon laser. Ann Plast Surg. 1987;18(1):47–50.
72. Stadler I, Lanzafame RJ, Evans R, Narayan V, Dailey
B, Buehner N, et al. 830-nm irradiation increases the
wound tensile strength in a diabetic murine model.
Lasers Surg Med. 2001;28(3):220–6.
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© Springer International Publishing AG, part of Springer Nature 2018
K. Nouri (ed.), Lasers in Dermatology and Medicine,
Lasers in Hair Growth and Hair
Transplantation
Nicole E. Rogers, Marc R. Avram,
Isabella Camacho, and Ali Rajabi-Estarabadi
Abstract
Results from hair transplantation are consis-
tently natural.
Demand for the procedure has increased
significantly.
Lasers have been implemented in the field
of hair transplantation to create recipient sites
and enhance hair growth.
Keywords
Hair growth · Hair transplantation · Lasers
• Results from hair transplantation are consis-
tently natural
• Demand for the procedure has increased
significantly
• Lasers have been implemented in the field of
hair transplantation to create recipient sites
and enhance hair growth
Introduction
Lasers have been an exciting new technology in
the field of hair loss and hair transplantation.
Since the theory of selective photothermolysis
was introduced in 1983 by Anderson and Parrish
[1], there has been a virtual explosion of laser
applications, ranging from hair removal to facial
rejuvenation to treatment of unwanted blood ves-
sels. One area that is still early in its development
is lasers for hair loss and hair transplantation.
This is especially exciting because a relatively
limited number of treatment options exist for
treating hair loss. FDA-approved medical treat-
ments for hair loss include topical minoxidil and
oral finasteride. The minoxidil 5% foam was
recently approved for once daily use in women.
Some women can benefit from birth control pills,
oral spironolactone or finasteride (off-label in
pre-menopausal
women
due
to
risks
of
teratogenicity).
The field of hair transplantation has under-
gone a significant transformation during the
N. E. Rogers (*)
Hair Restoration of the South, Metairie, LA, USA
M. R. Avram
Department of Dermatology, Weill Cornell Medical
Center, New York, NY, USA
I. Camacho
School of Medicine, Georgetown University,
Washington, DC, USA
A. Rajabi-Estarabadi
Department of Dermatology and Cutaneous Surgery,
University of Miami Miller School of Medicine,
Miami, FL, USA
20

352
last 10–15 years. With the advent of follicular
unit transplantation, patients achieve far better
results than they could with the original unnatu-
ral punch grafts (Figs.  20.1, 20.2, 20.3 and
20.4). This cosmetic revolution has resulted in
an ever-­increasing demand for the procedure.
According to the International Society of Hair
Restoration Surgery, practice census results
from 2015 estimated that about 358,109 surgi-
cal patients and 697,372 non-­surgical patients
were treated in 2014 worldwide for hair resto-
ration. In the United States, 112,409 surgical
hair restoration procedures were performed in
2014, compared to 88,304 surgical hair restora-
tion procedures performed in 2012, showing a
27% increase [2].
This chapter will focus on the two major laser
applications affecting the hair: (1) laser-assisted
creation of recipient sites in hair transplantation,
and (2) the use of low-level-laser therapy to
increase hair growth. Both applications have
faced considerable controversy within the laser
and hair transplant community. We attempt to
provide a balanced perspective of each so that
their roles may be fairly assessed and possibly
even implemented.
Fig. 20.1  Patient 1: before hair transplantation
Fig. 20.2  Patient 1: after 1000 grafts
Fig. 20.3  Patient 2: before hair transplantation
Fig. 20.4  Patient 2: after 900 grafts
N. E. Rogers et al.

353
Hair Transplantation
In addition to medical therapy, hair transplanta-
tion can provide even more dramatic and perma-
nent results. The field of hair transplantation has
evolved considerably over the last few decades as
large ‘pluggy’ punch grafts have been replaced
with tiny 1–4 hair follicular unit grafts. Nowadays
patients can expect very natural-looking results
(Fig.  20.5a, b). Hairs are harvested from the
occipital donor area either as an elliptical strip,
and then separated into individual grafts using
magnification, or they are manually or roboti-
cally harvested individually using 0.8–1  mm
punches (follicular unit extraction, Fig.  20.6).
Then, the grafts are placed back into pinpoint
slits created in the areas of thinning. The body’s
natural clotting factors act as a glue to hold the
grafts in place. Most hair transplant doctors use
steel blades or needles to create recipient sites.
Over a week’s time small scabs form around the
grafts, and after 2–6 weeks the grafts will enter a
resting telogen phase and shed. By 6–9 months
the hairs start growing in and by 12–18 months,
patients can expect their full results.
In using needles to create recipient sites, phy-
sicians face several challenges. The foremost
among these is hemostasis. As each new recipient
site is created, bleeding makes graft placement
difficult and time-consuming. Heavy drinkers or
patients on aspirin or blood-thinning supple-
ments may present even more of a challenge.
Grafts may pop out and require replacement.
This challenge prevents many interested derma-
tologic surgeons from entering the field.
Lasers in Hair Transplantation
• Recipient sites for hair transplantation are
usually made with needles
• Difficulties with hemostasis called for the use
of CO2 lasers for recipient site creation
• In 1996, the FDA approved lasers for use in
hair transplantation
a
b
Fig. 20.5  (a) Male patient before hair transplant surgery.
(b) Same patient 9 months after hair surgery (1050 1–4
hair grafts)
Fig. 20.6  Harvesting of single follicular units using
manual extraction process
20  Lasers in Hair Growth and Hair Transplantation

354
The first application of lasers in hair trans-
plant surgery was for the creation of recipient
sites in areas of thinning hair. CO2 lasers of the
10,600 nm wavelength were developed for abla-
tive resurfacing of the face and skin. They were
used to create incisions several millimeters deep
where the hair grafts could be placed. The goal
was to create recipient sites with better hemosta-
sis because the CO2 laser would stop bleeding
instantly [3]. Also because the alopecic skin
would be vaporized, there would be less risk of
graft compression and greater density (hairs per
square cm) could be achieved [4].
Carbon dioxide lasers were introduced in the
mid-1960s. They were popularized in the 1970s in
dermatologic surgery because of their ability to
vaporize tissue and seal blood vessels. The prob-
lem with continuous wave carbon dioxide lasers
was the peripheral non-specific spread of heat,
resulting in widespread scarring of the skin. In the
1990s, the concept of selective photothermolysis
was applied to carbon dioxide lasers. By limiting
the exposure time of the laser to less than one-half
of the thermal relaxation time of the surrounding
tissue, there was a significant reduction in lateral
thermal damage. This allowed CO2 lasers to
vaporize tissue with excellent hemostasis and no
scarring. Pulsed CO2 lasers were introduced to
treat dermatoheliosis and acne scarring with dra-
matic results.
The problems of bleeding and popping of
grafts at recipient sites remained. The investiga-
tion of handpieces for creating recipient sites in
pulsed CO2 lasers was begun in the mid-1990s to
over come this problem. High energy pulsed CO2
lasers would vaporize tissue and seal dermal ves-
sels with excellent hemostasis.
Initial studies found that average graft hair
counts were greater in laser-created sites in four
of the ten patients, and looked more natural [3].
They also found that there was less bleeding in
the laser-created sites, and that the associated
grafts required less handling. Despite extensive
research by highly experienced hair transplant
surgeons, the long-term implementation of
lasers for hair transplant surgery was limited.
This was because in some cases the complete
sealing off of blood led to limited graft ‘take’
and frequent graft fallout [5]. By adjusting the
laser’s energy down slightly, there was an
increase in “biological glue” that kept future
grafts from falling out.
Subsequent studies confirmed the ability to
make recipient sites with excellent hemostasis
and growth of transplanted hair (Figs. 20.7 and
20.8) [5, 6]. These studies led to FDA approval of
lasers for hair transplantation in 1996 [7–9]. FDA
approval resulted in a worldwide interest and use
of lasers to create recipient sites. Yet, today very
few hair surgeons use laser to make recipient
sites. What happened?
Fig. 20.7  Laser recipient sites versus steel created sites
Fig. 20.8  Physician making CO2 laser assisted recipient
sites
N. E. Rogers et al.

355
• Lasers
have
a
minor
role
in
hair
transplantation
• Steel needles can provide closer site creation,
with less lateral damage, resulting in greater
density and less post-surgical hemorrhagic
crusting
Some clinicians and investigators reported
superficial de-epithelialization surrounding the
sites. This led to greater and longer-lasting
crusts post-operatively. Delayed hair growth of
2–6  weeks was also observed [5]. The chief
obstacle over time was density. There was sig-
nificant collateral damage to the surrounding
tissues, which affected the growth of existing
pigmented terminal hair follicles. Histologic
studies of laser-­created site showed a minimum
of 20–50 μm of lateral thermal damage through
the dermis (Fig.  20.9). Although lasers were
able to create excellent hemostasis and growth
of transplanted hair, it did not allow for close
placement of recipient sites among existing hair
follicles. Nineteen gauge needles could more
safely create closely spaced sites than pulsed
CO2 lasers. If the recipient sites were created
too close together, poor growth, telogen efflu-
vium, or complete skin necrosis could occur.
Add to these problems the expense of the laser
purchase and maintenance, the risk of damage
to water-containing organs such as the cornea,
as well as the associated learning curve, and the
technique has largely fallen out of favor
[10–12].
To overcome the lateral tissue necrosis associ-
ated with CO2 lasers, clinicians have tried to use
holmium:YAG or erbium:YAG lasers instead of
traditional CO2 lasers for recipient site creation.
Histologic studies of the Ho:YAG (λ = 2120 nm)
demonstrated no advantage over traditional CO2
[4]. It created jagged, irregular-shaped channels
with even larger zones of thermal injury. The
Er:YAG (λ  =  2940  nm) has shown promising
results in creating recipient sites for both andro-
genetic alopecia [13] and cicatricial alopecia
[14]. Its use in a 2-year clinical trial produced
greater than 95% yield of 1–4-hair follicular units
with no reported side effects [15]. However, its
use has been limited by the lack of hemostasis
and the inability to create sites deep enough with
single pulse (Fig. 20.10).
There have been newer studies showing the ben-
efits of using ablative carbon dioxide (CO2) frac-
tional laser therapy. In one of the most recent
studies, it has been shown that combining hair
growth factors with ablative CO2 fractional laser
therapy is more beneficial than using hair growth
factor alone for male androgenetic alopecia (MAA).
After six treatment sessions involving 27 patients in
a randomized half-split study, 93% of patients in the
combined group showed improvement compared to
67% of patients in the growth factor group.
Discomfort due to treatment resolved in 2–3 days in
all patients, with the most common side effect being
post-treatment erythema [16]. Another study deter-
mined that ablative CO2 fractional lasers induce hair
Fig. 20.9  Pathology showing CO2 laser assisted recipient
sites for hair transplantation
Fig. 20.10  CO2 laser and Erbium laser
20  Lasers in Hair Growth and Hair Transplantation

356
growth by the activation of the Wnt10b/β-catenin
pathway in  vivo, with results indicating that the
10 mJ/spot and 300 spots/cm2 setting was the most
effective dosage [17]. According to this study, after
using an ablative CO2 fractional laser, the wound
healing process results in progression into the ana-
gen phase of the hair follicle cycle, thus, increasing
hair growth.
Using lasers for hair transplantation can be
helpful for novices to the field, in controlling
bleeding and reducing time to place the grafts.
However, it may take more time to create sites
with an appropriate angle and spacing.
Recipient sites cannot be made too close
together due to the dermal necrosis. If they are
too closely made, widespread necrosis of the
scalp may occur. It is unclear whether the
vaporization of tissue by laser creates enough
density
to
outweigh
this
requirement.
Ultimately, one must consider factors like the
cost of the laser and whether this technique is
superior to current standard of practice. It may
be hard for an expensive, complex laser to beat
the ease and economy of using needles or steel
blades, especially when bleeding is not a sig-
nificant problem for the experienced hair trans-
plant surgeon.
Perhaps novel fractional ablative devices will
be able to create recipient sites as close as #19
and #20 gauge needles without the increased risk
of necrosis. Creating a fractional ablative hand-
piece with a scanner would allow recipient sites
to be created quicker, with better hemostasis
leading to reduced operating time for patient and
physician.
• CO2 ablative lasers have FDA approval for
hair transplantation.
• Not used in hair transplants due to density.
• If laser transplant is performed, spacing too
close will result in telogen effluvium.
• Fractional ablative lasers may allow greater
density in the future.
Risks Inherent in CO2 Laser Usage
• Damage to water-containing organs, such as
the cornea
• Formation of smoke plume containing possi-
bly infectious disease
• Potential for igniting a fire with concomitant
oxygen use with sedation
There are also risks inherent in the CO2 laser
because it targets water as its primary chromo-
phore. Laser operators must be aware of the pos-
sibility of damaging organs high in water content,
such as the cornea. There is also the formation of
a smoke plume, which could contain bacteria,
viral DNA, or viable cells. The high voltage of a
CO2 laser may pose an electrical hazard by ignit-
ing tissue, oxygen, or volatile solvents used in
hair products. As seen in (Table 20.1), there are
as many advantages as there are disadvantages to
using lasers for hair transplantation.
More recent methods include robotic technol-
ogy that is now being used to harvest individual
follicular grafts from the back of the scalp, and
this may soon be applied to the creation of recip-
ient sites as well [23]. Table 20.2 includes a list
of different tools that presently used most fre-
quently for the creation of recipient sites
(Fig. 20.11).
Lasers in Hair Growth
• LLLT (Low Level Light Therapy) has been
further studied in randomized, controlled tri-
als showing beneficial results
• Devices are sold without a prescription,
through direct-to-consumer marketing
The application of lasers for hair growth is
based on the rare observation of hair growth in
patients after laser hair removal. This ‘paradoxi-
cal hypertrichosis’ has been reported following
treatment with the long-pulsed Nd:Yag laser
(755 nm) [24], the diode laser (810 nm) [25],
and with intense pulsed light (650–1200  nm)
[26–28]. Endre Mester, a Hungarian physician,
was the first to observe in the 1960s that mice
treated with lasers designed to prevent cancer
actually regrew hair in half the time of mice not
exposed to laser treatment [29]. He shaved off
their dorsal hair and divided the mice into
N. E. Rogers et al.

357
Table 20.1  Pros and cons of lasers in hair transplantation
Pros of lasers in hair transplantation
1. Improved hemostasis because blood vessels are sealed by laser
2. Reduced time to plant grafts
3. May be easier for beginner hair transplant doctors, as well as their assistants
4. Nerve endings, like blood vessels, are sealed with theoretical reduction in pain [3]
5. May achieve higher density because tissue is vaporized, not just pushed apart [4]
6. Less graft compression, again because alopecic skin measuring 1–2 mm wide is vaporized [3]
7. Less handling of the graft itself, because the laser-prepared sites are wider (1–2 mm) than scalpel created sites.
8. Don’t have to constantly sharpen 1–2 mm trephines [3]
9. Less risk of cobblestoning (from graft elevation) or pitting (from graft depression) because there is a constant
depth of laser-created site [3]
10. Use of less epinephrine [10]
11. Shorter operative time, which translates to less patient discomfort and less staff frustration [18]
12. Lower risk of cyst formation as a result of tissue left behind [19]
Cons of lasers in hair transplantation
1. Expense of laser, required office space, and maintenance [20, 21]
2. May fray epithelial edges and cause superficial de-epithelialization [5]
3. Greater postoperative crusting [4, 22]
4. Delayed (2–6 weeks) regrowth of transplanted follicles [5]
5. Risk of telogen effluvium from heat of laser—may thin sites with existing hair
6. Increased risk of secondary infection [5]
7. Risk of injury to adjacent hair follicles in recipient area [5]
8. May achieve lower density because cannot place so close as to risk necrosis [20, 21]
9. Longer operating time, because of difficulty creating site at the appropriate angle [5]
10. Persistent erythema [22]
11. Learning curve associated with using laser [5]
12. Plume may contain noxious and carcinogenic chemicals
Fig. 20.11  Instruments presently used to create recipient
sites
control and treatment groups, the latter receiv-
ing a low-powered ruby laser therapy (694 nm).
He found no evidence of cancer in the mice, but
did observe that the laser-treated group had
faster hair regrowth. This was the origin of “bio-
stimulation” using “cold laser” or “soft laser”
therapy administered at lower powers of
1–500  mW.  Higher powered lasers, emitting
1–10  W, are used to clear blood vessels or
hyperpigmentation.
Several other parameters are involved in
administering LLLT.  Wavelengths of 600–
700 nm are used to treat superficial tissues, while
780–950 nm wavelengths are used for deeper tis-
sues. There is a biphasic dose response curve, in
which the central distribution, 700–770 nm is not
considered to have as much activity. The dose of
energy is comparable to regular laser use,
between 1 and 20 J, but it is delivered in a much
slower way (Power = J/s = watts).
Basic Science of LLLT
Table 20.2  Traditional tools for recipient site creation
Traditional needles: 17 g (4 hair FU’s), 18 g (3 hair
FU’s), 19 g (2 hair FU’s) or 20 g (1 hair FU’s)
Chisel blades (custom cut)
Spear point blade
Minde blade
No-core needles
20  Lasers in Hair Growth and Hair Transplantation

358
• Stimulates the mitochondrial transport chain
• Enhances ATP production
• Stimulates wound healing
• Reduces inflammation
• Improves neurologic damage, such as with
stroke
• Improves musculoskeletal and joint pain
The use of LLLT is based on several scientific
papers showing that it can increase ATP levels in
tissues by stimulating the mitochondrial transport
system [30–32]. To fully understand this we must
briefly review the structure and mechanisms of
ATP synthesis in mitochondria. These intracellu-
lar organelles are considered to be the power-
houses of the cell. They have an outer membrane
and an inner membrane, which has numerous
infoldings or cristae. Between these two mem-
branes there is an intramembranous space. The
very center of the mitochondria is called the
matrix (Fig. 20.12).
The respiratory chain has five major com-
plexes that shuttle electrons from the intramem-
branous space into the matrix. These include
NADH dehydrogenase (Complex I), ATP succi-
nate dehydrogenase (Complex II), cytochrome c
reductase (Complex III), cytochrome c oxidase
(Complex IV), ATP synthase (Complex V) and
two freely diffusible molecules ubiquinone and
cytochrome c that shuttle electrons from one
complex to the next [33]. By transferring elec-
trons centrally, a proton gradient is built up in the
intermembranous space. These protons enter
back into the mitochondrial matrix through chan-
nels in the ATP synthase enzyme complex. This
entry is coupled to ATP synthesis from ADP and
phosphate (Pi) (Fig. 20.13).
LLLT has been found to increase the activity
of Complex II and Complex IV in particular [34].
This was demonstrated in a controlled study of
wounds treated with AsGa (gallium arsenate,
904 nm) low-level laser. This study also showed
a clinical improvement in the rate of wound heal-
ing after LLLT.  Another study using the same
laser but at a slightly different wavelength
(808 nm) showed enhancement of ATP ­production
in human neuronal cells in culture [30]. This sup-
ports the observation that LLLT can help in the
setting of neurologic damage following strokes
[35, 36]. Its ability to repair neurologically dam-
aged tissue may be a function of inhibiting nitric
oxide synthase and upregulating the expression
of transforming growth factor-beta 1 [37].
It appears that low-level-lasers can help not
only with neurologic damage but also with neu-
rogenic pain and musculoskeletal complaints.
Peer-reviewed studies found it helpful in treating
low back pain [38], temporomandibular joint dis-
orders [39], and rheumatoid arthritis patients
with carpal tunnel syndrome [40]. In fact, the
MicroLight 830 is a low level laser, which
received 510K medical device clearance by the
FDA in 2002 for the treatment of carpal tunnel
syndrome. Some chiropractors and practitioners
of holistic medicine attest to its usefulness in
treating patients with other chronic disorders like
fibromyalgia [41, 42]. They liken it to acupunc-
ture, in providing a noninvasive treatment where
other options have failed. As mentioned above,
LLLT can improve wound healing.
One study showed that the helium neon laser,
at a wavelength of 633 nm, was effective in stim-
ulating the cellular responses of wounded fibro-
blasts and promoting cell migration [43]. Another
study looking at LLLT for wound healing in dia-
betic rats found faster healing in the treatment
group, again with an optimum wavelength of
633 nm [44].
Perhaps most important is the evidence that
LLLT can reduce levels of inflammation in the
Outer membrane
Intramembranous space
Inner membrane
Matrix
*
Cristae
*
Fig. 20.12  Structure of mitochondria
N. E. Rogers et al.

359
tissues. It was found to reduce the levels of TNF-­
alpha in rats treated with a 650  nm  Ga-Al-As
laser [45]. It has also been found to reduce levels
of serum prostaglandin E2 in rats with zymosan-­
induced arthritis that underwent illumination
with 810 nm laser [46]. The authors also observed
a reduction in joint swelling that was comparable
to treatment with dexamethasone. Finally, levels
of COX-2 mRNA expression are also reduced in
patients treated with LLLT [47].
These results suggest that LLLT may be use-
ful in treating autoimmune and other disorders
based on inflammation, where primary treat-
ments have failed. Hair disorders such as lichen
planopilaris and alopecia areata may even bene-
fit. Androgenetic alopecia, which is not an
inflammatory process, may improve more from
increased energy and ATP created by the laser.
Studies are lacking to directly link the production
of ATP with the enhancement of hair growth.
However, there are many drugs whose mecha-
nism of action is still unclear. It would be a shame
to omit a helpful treatment from our armamen-
tarium simply because we do not yet understand
it. Hopefully more studies will soon bring this to
light.
One possible explanation for the effect of light
on hair follicles is photo-relaxation, a concept
proposed by Dr. Robert Furchgott in the 1950s
[48]. He observed that strips of smooth muscle
from intestinal tissue would alternately contract
and relax as he cast a shadow or allowed UV light
to shine on it, respectively. Years later he won the
Nobel prize for identifying that UV light acti-
vates the release of nitric oxide from vascular
smooth muscle cells [49]. This photo-activated
vasodilation may allow increased blood supply to
nearby hair follicles. If so, this would be similar
to minoxidil’s proposed effect of vasodilation on
the follicles.
LLLT Products
The application of low-level light therapy (LLLT)
for male and female hair loss began shortly after
the turn of the century with only manufacturer
data. A laser comb delivery of LLLT was FDA
cleared for treatment of male pattern hair loss in
2007, but with mixed reviews among physicians
[22, 50]. The devices were marketed directly to
consumers via the internet, television, and other
print advertisements. Soon, however, hair loss
specialists began to offer chair-type devices in
their offices and sold helmets, hats, and brushes
and combs that contained the low-level light
technology. In 2011 the same laser comb device
was cleared for treatment of female pattern hair
Intermembrane
space
+
e
QH2
Q
+
+
I
II
III
IV
+
+
e
e
+
+
+
+
+
+
+
+
+
4H+
2H+
H+
Cyto
Cyto
Fumarate
Succinate
Mitochondrial
matrix
H2O
ATP
ADP+Pi
1/2O2+2e-
NAD-
NADH2
C2+
C3+
4H+
Fig. 20.13  Structure of the mitochondrial transport chain (Reprinted from Hamblin MR, Demidova TN. Mechanisms
of low level light therapy. Proc SPIE. 2006;6140:614001 [33], with permission from the SPIE)
20  Lasers in Hair Growth and Hair Transplantation

360
loss as well [51]. Since the last edition of this
book, there has been a further increase in the
number of commercially available LLLT devices
(Table 20.3).
The Hairmax Lasercomb™ has been one of
the most publicized products on the market. It
was developed and patented by Lexington
International in 2000. Although the exact
wavelength and other parameters are kept con-
fidential, the manufacturers do reveal that it
uses a diode laser operating in the red portion
of the visible color spectrum [52]. It gives off a
monochromatic, collimated laser energy. It is
indicated to promote hair growth in males with
androgenetic alopecia who have Norwood
Hamilton Classifications of Ia to V and
Fitzpatrick skin types I to IV.  Reports of the
data they submitted to the FDA are positive,
showing increased hair counts among almost
all patients in their four-site study. However,
they have chosen to keep the specifics of this
data proprietary. And although the trials were
done only in men, they market the product to
women as well. The FDA approved it in
January 2007 after the company filed a 510(k)
notice, requesting that it be registered as a
medical device.
One caveat is that FDA approval of medical
devices is far less rigorous than it is for standard
pharmaceutical drugs. This labeling indicates
that the FDA has reviewed the product and found
it to be safe and ‘substantially equivalent’ to
predicate devices already on the market. The
most similar predicate device is the TerraQuant, a
handheld LLLT device emitting 60–90 W within
the 600–900  nm wavelength for treatment of
musculoskeletal pain [53]. Other such devices
include the Violet Ray device, which was manu-
factured in the 1950s as a treatment for nearly
everything but later its makers were charged with
libel and misbranding [54].
Some other names in the industry of LLLT for
hair growth are Sunetics® International, located
in Las Vegas, NV [55] and the Revage 670® laser
[56]. The Sunetics product comes either as a
brush ($200–400) or as a freestanding machine
for total scalp treatments ($39,900). It uses a
650 nm wavelength at a fluence of 5 mW. It is
marketed especially among hair transplant offices
to increase the quality/quantity of the donor area,
to reduce the pain and to speed wound healing
after transplant, and to prevent or reduce hair loss
from post-transplant shock. It also is sold as an
option for men and women who do not want to
undergo a hair transplant procedure and haven’t
improved with minoxidil or finasteride. The
Revage 670® is a chair-based laser that uses rota-
tional phototherapy containing 30 diodes that
rotate 180° around the scalp. It is given as in-­
office treatments 2×/week for 6 weeks then once
weekly for 16 weeks. Each treatment session is
30 min each.
The first study investigating low-level light
therapy for hair loss was published in 2003 in a
non-peer-reviewed journal [57]. It was sup-
ported by the manufacturers of HairMax
LaserComb™. It enrolled 35 patients with
androgenetic alopecia (AGA). Twenty-eight
males and seven females were given a handheld
laser comb to use at home for 6 full months,
combing the hair for 5–10 min daily. They found
that overall, for men and women there was a
93.5% increase in hair counts in both temporal
and vertex sites. Tensile strength of individual
hairs also increased by 78.9%.
Another independent study was performed by
the authors in 2008 using the Sunetics® clinical
unit (hood) device. Participants received 20-min
treatment sessions twice weekly for 3–6 months.
Trichoscopy showed a decrease in the number of
vellus hairs, increase in the number of terminal
hairs, and an increase in shaft diameter, however
the study was limited by a small patient popula-
tion (n = 7) and the results were not statistically
significant [58].
In 2009, the first of several randomized con-
trolled trials was published. It was supported by
Table 20.3  Low-level light therapy devices
Hairmax LaserComb™
Sunetics® Laser Hair Brush and Clinical unit
Revage® 670 laser (chair unit)
Spencer Forrest X5 (Handheld) Hair Laser™
LaserCap® (via physicians only)
iGrow® Hair laser System
Capillus® (via physicians only)
N. E. Rogers et al.

361
the manufacturers of HairMax LaserComb™.
This was a multi-center sham-device controlled
study that did demonstrate statistically significant
improvements in hair growth in men over a sham
device [59].
In 2013, several more randomized, controlled
trials were published. One study tested the Oaze,
a helmet-type device emitting LEDs with wave-
lengths of 630, 650 and 660 nm. Forty Korean
subjects with AGA were enrolled and received
either an active or sham device to use at home for
18 min daily. A tattoo was used to mark the area
of study within the frontal or vertex area, and
phototrichogram analysis was performed at base-
line, 12 weeks, and 24 weeks. After 24 weeks of
treatment, the active group showed statistically
significantly greater hair density than the sham-­
device group.
Another
randomized,
placebo-controlled
trial was performed in 2013 also using a helmet-
type device called the “TOPHAT655” (investi-
gational version of the iGrow [60]). It contained
twenty-­one 5 mW lasers (655 ± 5 nm), and 30
LEDs (655  ±  20  nm). Forty-four males (age
18–48) with androgenetic alopecia were
enrolled and 41 completed the study (22 active,
19 placebo). Areas of thinning were trimmed to
3 mm, tattooed, and photographed. The control
group was given an identical device with incan-
descent lights that were painted red. Patients
used the devices at home for 25 min per treat-
ment on alternating days for 16 weeks (total of
60 treatments). Statistical analysis was blinded.
After deleting one outlier (placebo group) data
point, the investigators found a 35% increase in
the number of hairs in the treatment group com-
pared with the sham-device group (P = 0.003).
No distinction was made between vellus and ter-
minal hairs, and the diameter of the hairs was
not measured. No females were included in the
study, but according to the author a second man-
uscript showing similar results for women is in
press [61].
A very recent study was a second multi-cen-
ter, randomized, sham-device controlled double-­
blind study using the HairMax Lasercomb™
[62]. The authors included well-respected
experts in hair loss at major academic
institutions. All trials were registered at www.
clinicaltrials.gov as well as the Institutional
Review Boards for each participating author. A
total of 128 male and 141 female subjects with
AGA were randomized to receive either a laser
comb (1 of 3 models) or a sham device (emitting
white light), to apply to the scalp three times
weekly for 26  weeks. The subjects and site
investigators remained blinded as to the type of
device and the analysis of the digital photo-
graphs was also blinded.
Overall, 103 males and 122 females com-
pleted the study. The female patients had an
increase in mean terminal hair count from base-
line of 20.2, 20.6, and 18.4 hairs per cm2 in the
9-beam, 12-beam, 7-beam devices compared
with 2.9 (p  <  0.0001), 3.0 (p  <  0.0001), 1.6
(p = 0.0017) hairs/cm2 in sham-treated patients.
Among male patients, the mean terminal hair
count increased by 20.9 and 25.7 hairs per cm2
for the 9- and 12-beam devices compared with
9.4 (p = 0.0249) and 9.4 (p = 0.0028) hairs per
cm2 with the sham-devices. Also, a higher per-
centage of the patients in the active treatment
group reported improvement in overall hair loss
condition, thickness, and fullness in self-­
assessment than did the sham-treated individu-
als (Figs. 20.14 and 20.15).
LLLT Mechanisms of Action for Hair
Growth in Nutshell
Originally, LLLT was hypothesized to grow
hair by increasing mitochondrial signaling to
ultimately increase ATP levels in tissues [32,
33, 63]. Since then, it has been suggested that
LLLT has similar (but not clearly delineated)
effects as minoxidil on the hair follicle [64].
These mechanistic overlaps may include a
release of nitric oxide, [65] localized vasodila-
tion [66], or the opening of potassium channels
[67]. LLLT may also work by activating NF-kB
signaling with subsequent anti-apoptotic effects
[68]. There is fairly good evidence that LLLT
can increase levels of vascular endothelial
growth factor (VEGF) resulting in more rapid
angiogenesis [69–71]. It appears to do so via
20  Lasers in Hair Growth and Hair Transplantation

362
the ERK/Sp1 signally pathway [72]. Whether
this has more of an effect on hair growth or
wound healing is uncertain.
Recently, the role of LLLT in hair growth
was evaluated in C3H/HeJ mice, leading to the
conclusion that LLLT induces the anagen phase
of hair follicles, thus promoting hair regrowth
by initiating the Wnt10b/β-catenin pathway
[73]. In this study, the LLLT group expressed a
significantly higher level of Wnt10b and
β-catenin on the second day than the control
group according to both the western blot and
PCR results. Wnt signaling, especially Wnt10b
expression, is necessary for hair follicle devel-
opment by activating bulge stem cells towards
hair formation [74, 75]. Activation of the
Wnt10b/β-catenin pathway has been seen to
result in regeneration of hair follicles [76].
Based on a recent evidence-based review on
LLLT devices for treating alopecia, it was found
that FDA-cleared devices are effective for
patients with pattern hair loss, with low inci-
dence of adverse effects [77].
Fig. 20.14  Clinical results before and 26  weeks after treatment with HairMax Lasercomb™ (photos courtesy of
Lexington International, LLC)
Fig. 20.15  Microscopic results before and 26  weeks
after treatment with HairMax Lasercomb™ (photos cour-
tesy of Lexington International, LLC)
N. E. Rogers et al.

363
Lasers for Wound Healing
There have been a significant number of publica-
tions investigating the role of LLLT for treating
wounds. Most of it has been limited to in vitro or
mouse models, and do not thoroughly differenti-
ate between photothermal, photochemical, or
photomechanical effects [78]. A 635  nm laser
was found to stimulate wound contraction in sus-
ceptible mouse strains [79]. It was found to
increase levels of basic fibroblast growth factor
(bFGF) and insulin-like growth factor-1 (IGF-1)
with greater statistical significance than was seen
in control groups [80]. Based on these studies,
LLLT may have a role in the post-operative
period following hair transplantation. Most
patients experience scabbing over the grafted
area that can last for 1–2  weeks after surgery.
They may also have discomfort in the donor area
for 1–3 days after surgery. The use of LLLT in
treating the grafted and/or donor areas after hair
transplantation may be an area of future study.
More human clinical trials are needed.
Conclusion
• The use of lasers in hair growth and trans-
plantation remains controversial
• Needles remain the standard of treatment
for transplant site creation, due to effi-
ciency, economy, and improved density
• FDA approval of lasers for hair growth
suggests that they are safe
• More well-designed clinical trials are
needed to assess their efficacy
While the use of lasers to create recipient
sites in hair transplantation has largely fallen
out of favor, lasers may still have a role in
medical therapy for hair loss, as well as
improving post-­operative wound healing. The
volume of medical literature supporting the
role of LLLT for both is still increasing, and
with more evidence of statistically significant
results, the implementation of lasers may
become more prevalent. That being said, con-
sumers can purchase a lot of topical minoxidil
or oral finasteride for the pricey cost of these
lasers. And, many argue that if the improve-
ment can only be detected microscopically
then it may not be enough to warrant its wide-
spread use. Presently, the authors recommend
using laser therapy as an adjunct to medical
therapy if optimal results are not happening
with minoxidil and/or finasteride.
No matter how we implement lasers to treat
patients with hair loss, we must first identify
the etiology. Frequently conditions such as
lichen planopilaris or alopecia areata may
present in a way that mimics androgenetic alo-
pecia. We should be sure that the patients
undergo medical evaluation and biopsy where
necessary. This crucial step may be left out
when treatments such as LLLT are available
directly to the public without a prescription.
Consumers should be protected from buying
expensive items that may not be applicable or
aggressive enough for their type of hair loss.
Likewise, physicians should be open to the
use of such devices where other options have
failed, so long as reproducible studies can
demonstrate their safety and efficacy.
References
1. Anderson RR, Parrish JA. Selective photothermoly-
sis: precise microsurgery by selective absorption of
pulsed radiation. Science. 1983;220:524–7.
2.
report_2015_practice_census_final_v2.pdf
3. Unger WP, David LM. Laser hair transplantation. J
Dermatol Surg Oncol. 1994;20:515–21.
4. Chu EA, Rabinov CR, Wong BJF, Krugman
ME.  Laser-assisted hair transplantation: histo-
logic comparison between CO2 and Ho:YAG lasers.
Dermatol Surg. 2001;27:335–42.
5. Unger WP.  Laser hair transplantation II.  Dermatol
Surg. 1995;21:759–65.
6. Unger WP.  Laser hair transplantation III.  Dermatol
Surg. 1995;21:1047–55.
7. Grevelink JM, Brennick JB.  Hair transplantation
facilitated by flashscanner-enhanced carbon dioxide
laser. Otolaryngol Head Neck Surg. 1994;5:278–80.
8. Grevelink JM.  Laser hair transplantation. Dermatol
Clin. 1997;15:479–86.
9. Villnow M. 2300 grafts/laser session. Hair Transplant
Forum. 1994;4:6–7.
10. Grevelink JM, Farinelli W, Bua D, et al. Hair trans-
plantation aided by CO2 lasers. Lasers Surg Med.
1995;7:47.
11. Smithdeal CD. Carbon dioxide laser-assisted hair trans-
plantation: the effects of laser parameters on scalp tis-
sue – a histologic study. Dermatol Surg. 1997;23:835–40.
20  Lasers in Hair Growth and Hair Transplantation

364
12. Kauvar AN, Waldorf HA, Geronemus RG. A histo-
pathological comparison of “char-free” carbon diox-
ide lasers. Dermatol Surg. 1996;22:343–8.
13. Neidel FG, Fuchs M, Krahl D. Laser-assisted autolo-
gous hair transplantation with the Er:YAG laser. J
Cutan Laser Ther. 1999;1:229–31.
14. Podda M, Spieth K, Kaufmann R.  Er:YAG laser-­
assisted hair transplantation in cicatricial alopecia.
Dermatol Surg. 2000;26:1010–4.
15. Uebel C. The use of erbium:YAG laser in hair micro-­
transplant surgery. Clin Appl Notes. 1999;7:1.
16. Huang Y, Zhuo F, Li L. Enhancing hair growth in male
androgenetic alopecia by a combination of fractional
CO2 laser therapy and hair growth factors. Lasers Med
Sci. 2017;32:1711.
17. Bae JM, Jung HM, Goo B, Park YM. Hair regrowth
through wound healing process after ablative frac-
tional laser treatment in a murine model. Lasers Surg
Med. 2015;47(5):433–40.
18. Ho C, Nguyen Q, Lask G, Lowe N.  Mini-slit hair
transplantation using the Ultrapulse carbon dioxide
laser handpiece. Dermatol Surg. 1995;21:1056–9.
19. Unger WP. What’s new in hair replacement surgery.
Dermatol Clin. 1996;14:783–802.
20. Avram MR.  Laser-assisted hair transplantation  – a
status report in the 21st century. J Cosmet Dermatol.
2005;4:135–9.
21. Avram MR, Leonard RT Jr, Epstein ES, Williams JL,
Bauman AJ. The current role of laser/light sources in
the treatment of male and female pattern hair loss. J
Cosmet Laser Ther. 2007;9:27–8.
22. Fitzpatrick RE. Laser hair transplantation. Tissue effects
of laser parameters. Dermatol Surg. 1995;21:1042–6.
23. Rose PT, Nusbaum B.  Robotic hair restoration.
Dermatol Clin. 2014;32:97–107.
24. Alajlan A, Shapiro J, Rivers JK, MacDonal N, Wiggin
J, Lui H. Paradoxical hypertrichosis after laser epila-
tion. J Am Acad Dermatol. 2005;53:85–8.
25. Bernstein EF.  Hair growth induced by diode laser
treatment. Dermatol Surg. 2005;31:584–6.
26. Willey A, Torrontegui J, Azpiazu J, et al. Hair stimu-
lation following laser and intense pulsed light photo-­
epilation: review of 543 cases and ways to manage it.
Lasers Surg Med. 2007;39:297–301.
27. Moreno-Arias G, Castelo-Branco C, Ferrando
J.  Paradoxical effect after IPL photoepilation.
Dermatol Surg. 2002;28:1013–6.
28. Radmanesh M. Paradoxical hypertrichosis and termi-
nal hair change after intense pulsed light hair removal
therapy. J Dermatol Treat. 2009;20:52–4.
29. Mester E, Szende B, Gartner P.  The effect of laser
beams on the growth of hair in mice. Radiobiol
Radiother (Berl). 1968;9:621–6.
30. Oron U, Ilic S, De Taboada L, Streeter J.  Ga-As
(808 nm) laser irradiation enhances ATP production
in human neuronal cells in culture. Photomed Laser
Surg. 2007;25:180–2.
31. Gavish L, Asher Y, Becker Y, Kleinman Y. Low level
laser irradiation stimulates mitochondrial membrane
potential and disperses subnuclear promyelocytic leu-
kemia protein. Lasers Surg Med. 2004;35:369–76.
32. Yu W, Naim JO, McGowan M, Ippolito K, Lanzafame
RJ.  Photomodulation of oxidative metabolism and
electron chain enzymes in rat liver mitochondria.
Photochem Photobiol. 1997;66:866–71.
33. Hamblin MR, Demidova TN.  Mechanisms of low
level light therapy. Proc SPIE. 2006;6140:614001.
34. Silveira PC, Streck EL, Pinho RA. Evaluation of mito-
chondrial respiratory chain activity in wound healing
by low-level laser therapy. J Photochem Photobiol B.
2007;86:279–82.
35. Oron A, Oron U, Chen J, et al. Low-level laser therapy
applied transcranially to rats after induction of stroke
significantly reduces long-term neurological deficits.
Stroke. 2006;37:2620–4.
36. Lampl Y, Zivin JA, Fisher M, et  al. Infrared laser
therapy for ischemic stroke: a new treatment strategy:
results of the NeuroThera Effectiveness and Safety
Trial-1 (NEST-1). Stroke. 2007;38:1843–9.
37. Leung MC, Lo SC, Siu FK, So KF.  Treatment of
experimentally induced transient cerebral ischemia
with low energy laser inhibits nitric oxide synthase
activity and up-regulates the expression of trans-
forming growth factor-beta 1. Lasers Surg Med.
2002;31:283–8.
38. Djavid GE, Mahrdad R, Ghasemi M, Hasan-Zadeh
H, Sotoodeh-Manesh A, Pouryaghoub G. In chronic
low back pain, low level laser therapy combined with
exercise is more beneficial than exercise along in
the long term: a randomized trial. Aust J Physiother.
2007;53:155–60.
39. Fikackova H, Dostalova T, Navratil L, Klaschka
J.  Effectiveness of low-level laser therapy in tem-
poromandibular joint disorders: a placebo-controlled
study. Photomed Laser Surg. 2007;25:297–303.
40. Ekim A, Armagan O, Tascioglu F, Oner C, Colak
M.  Effect of low level laser therapy in rheumatoid
arthritis patients with carpal tunnel syndrome. Swiss
Med Wkly. 2007;137:347–52.
41.
42.
43. Hawkins D, Abrahamse H.  Effect of multiple
exposures of low-level laser therapy on the cellu-
lar responses of wounded human skin fibroblasts.
Photomed Laser Surg. 2006;24:705–14.
44. Al-Watban FA, Zhang XY, Andres BL.  Low-level
laser therapy enhances wound healing in diabetic
rats: a comparison of different lasers. Photomed Laser
Surg. 2007;25:72–7.
45. Aimbire F, Albertini R, Pacheco MT, et al. Low-level
laser therapy induces dose-dependent reduction of
TNF-alpha levels in acute inflammation. Photomed
Laser Surg. 2006;24:33–7.
46. Castano AP, Dai T, Yaroslavsky I, et  al. Low-level
laser therapy for zymosan-induced arthritis in rats:
importance of illumination time. Lasers Surg Med.
2007;39:543–50.
47. Albertini R, Aimbire F, Villaverde AB, Silva JA Jr,
Costa MS.  COX-2 mRNA expression decreases
in the subplantar muscle of rat paw subjected to
carrageenan-­induced inflammation after low-level
laser therapy. Inflamm Res. 2007;56:228–9.
N. E. Rogers et al.

365
48. Ehrreich SJ, Furchgott RF. Relaxation of mammalian
smooth muscles by visible and ultraviolet radiation.
Nature. 1968;218:682–4.
49. Furchgott
RF,
Jothianandan
D. 
Endothelium-­
dependent and independent vasodilation involving
cyclic GMP: relaxation induced by nitric oxide, carbon
monoxide, and light. Blood Vessels. 1991;28:52–61.
50. Woolston C. Does Lasercomb stop hair loss? The evi-
dence is thin. Los Angeles Times. January 12, 2009;
51. Website:
fda-clearance/
52.
53.
54.
55.
56.
57. Satino JL, Markou M. Hair regrowth and increased
hair tensile strength using the HairMax LaserComb
for low-level laser therapy. Int J Cosmet Surg Aesthet
Dermatol. 2003;5:113–7.
58. Avram MR, Rogers NE.  The use of low-level
light for hair growth: part I.  J Cosmet Laser Ther.
2009;11:110–7.
59. Leavitt M, Charles G, Heyman E, Michaels
D.  HairMax LaserComb Laser Phototherapy device
in the treatment of male androgenetic alopecia. Clin
Drug Invest. 2009;29:283–92.
60. Lanzafame RJ, Blanche RR, Bodian AB, Chiacchierini
RP, Fernandez-Obregon A, Kazmirek ER. The growth
of human scalp hair mediated by visible red light
laser and LED sources in males. Lasers Surg Med.
2013;45:487–95.
61. Personal email communication with Dr. Raymond
Lanzafame on August 7, 2014.
62. Jimenez JJ, Wikramanayake TC, Bergfeld W, et  al.
Efficacy and safety of a low-level laser device in the
treatment of male and female pattern hair loss: a mul-
ticenter, randomized, sham device-controlled, double-­
blind study. Am J Clin Dermatol. 2014;15(2):115–27.
63. Karu TI. Mitochondrial signaling in mammalian cells
activated by red and near-IR radiation. Photochem
Photobiol. 2008;84:1091–9.
64. Avci P, Gupta GK, Clark J, Wikonkal N, Hamblin
MR. Low-level laser (light) therapy (LLLT) for treat-
ment of hair loss. Lasers Surg Med. 2014;46:144–51.
65. Karu TI, Pyatibrat LV, Afanasyeva NI. Cellular effects
of low power laser therapy can be mediated by nitric
oxide. Lasers Surg Med. 2005;36:307–14.
66. Proctor PH. Endothelium-derived relaxing factor and
minoxidil: active mechanisms in hair growth. Arch
Dermatol. 1989;125:1146.
67. Rossi A, Cantisani C, Melis L, Iorio A, Scali E,
Calvieri S. Minoxidil use in dermatology, side effects
and recent patents. Recent Patents Inflamm Allergy
Drug Discov. 2012;6:130–6.
68. Chen AC, Arany PR, Huang YY, Tomkinson EM,
Shamra SK, Kharkwal GB, Saleem T, Mooney D, Yull
FE, Blackwell TS, Hamblin MR. Low-level laser ther-
apy activates NF-kB via generation of reactive oxygen
species in mouse embryonic fibroblasts. PLoS One.
2011;6:e22453.
69. Cury V, Moretti AI, Assis L, Bossini P, Crusca Jde
S, Neto CB, Fangel R, DeSouza HP, Hamblin MR,
Parizotto NA.  Low level laser therapy increases
angiogenesis in a model of ischemic skin flap in rats
mediated by VEGF, HIF-1a and MMP-2. J Photochem
Photobiol B. 2013;125:164–70.
70. Zaidi M, Krolikowski JG, Jones DW, Pritchard KA
Jr, Struve J, Nandedkar SD, Lohr NL, Pagel PS,
Weihrauch D.  Transient repetitive exposure to low
level light therapy enhances collateral blood ves-
sel growth in the ischemic hindlimb of the tight skin
mouse. Photochem Photobiol. 2013;89:709–13.
71. Prado RP, Garcia SB, Thomazini JA, Piccinato
CE.  Effects of 830 and 670  nm laser on viability
of random skin flap in rats. Photomed Laser Surg.
2012;30:418–24.
72. Feng J, Zhang Y, Xing D. Low-power laser irradia-
tion (LPLI) promotes VEGF expression and vascular
endothelial cell proliferation through the activation of
ERK/Sp1 pathway. Cell Signal. 2012;24:1116–25.
73. Zhang T, Liu L, Fan J, et al. Low-level laser treat-
ment stimulates hair growth via upregulating Wnt10b
and beta-catenin expression in C3H/HeJ mice. Lasers
Med Sci. 2017;32(5):1189–95.
74. Andl T, Reddy ST, Gaddapara T, Millar SE.  WNT
signals are required for the initiation of hair follicle
development. Dev Cell. 2002;2(5):643–53.
75. Jang SK, Kim ST, Lee DI, et al. Decoction and fer-
mentation of selected medicinal herbs promote hair
regrowth by inducing hair follicle growth in conjunc-
tion with Wnts signaling. Evid Based Complement
Alternat Med. 2016;2016:4541580.
76. Li Y-H, Zhang K, Yang K, et al. Adenovirus-mediated
Wnt10b overexpression induces hair follicle regen-
eration. J Investig Dermatol. 2013;133(1):42–8.
77. Zarei M, Wikramanayake TC, Falto-Aizpurua L,
Schachner LA, Jimenez JJ. Low level laser therapy
and hair regrowth: an evidence-based review. Lasers
Med Sci. 2016;31(2):363–71.
78. Posten W, Wrone DA, Dover JS, Arndt KA, Silapunt
S, Alam M.  Low-level laser therapy for wound
healing: mechanism and efficacy. Dermatol Surg.
2005;31:334–40.
79. Demidova-Rice TN, Salomatina EV, Yaroslavsky AN,
Herman IM, Hamblin MR. Low-level light stimulates
excisional wound healing in mice. Lasers Surg Med.
2007;39:706–15.
80. Saygun I, Karacay S, Serdar M, Ural AU, Sencimen
M, Kurtis B. Effects of laser irradiation on the release
of basic fibroblast growth factor (bFGF), insulin
like growth factor-1 (IGF-1), and receptor of IGF-1
(IGFBP3) from gingival fibroblasts. Lasers Med Sci.
2008;23:211–5.
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367
© Springer International Publishing AG, part of Springer Nature 2018
K. Nouri (ed.), Lasers in Dermatology and Medicine,
Photobiomodulation and Hair
Growth
Molly B. Hirt and Ronda S. Farah
Abstract
Since the initial Food and Drug Administration
clearance of the first photobiomodulation
device for androgenetic alopecia in 2007, the
market for these devices has rapidly expanded.
Sixteen unique devices are currently available
to consumers with varying designs, treatment
durations and frequency. While the precise
mechanism for hair growth stimulation
remains to be elucidated, current evidence
suggests the laser light alters hair cycle dura-
tion to promote the anagen growth phase.
Photobiomodulation devices have an excellent
safety profile with pruritus and skin dryness
reported to be the most common side effects.
Overall, research has demonstrated clinical
efficacy of these devices, including random-
ized controlled studies. As photobiomodula-
tion continues to emerge as a treatment
modality for androgenetic alopecia, additional
information on the most effective light
sources, precise light wavelength, treatment
schedule, and effectiveness on various hair
diseases is still needed.
Keywords
Photobiomodulation (PBM) · Hair growth ·
Alopecia · Androgenetic alopecia (AGA) ·
Laser · Low-level laser therapy (LLLT) ·
Light-emitting diodes
Abbreviations
FDA
Food and Drug Administration
LED
Light-emitting diodes
mW
Milliwatts
nm
Nanometer
PBM Photobiomodulation
PRP
Platelet rich plasma
RCT
Randomized control trial
UBM Ultrasound bio-macroscopic
Background
Photobiomodulation (PBM) involves the use of
low-level red or near infrared light to stimulate a
photochemical reaction. The treatment of tissue
with low energy light, resulting in a therapeutic
response is termed PBM therapy [1, 2]. This type
of therapy has also been referred to as low level
laser (light) therapy or photobiostimulation
M. B. Hirt · R. S. Farah (*)
Department of Dermatology, University of
Minnesota, Minneapolis, MN, USA
e-mail: mbhirt@iupui.edu, hirtx018@umn.edu;
rfarah@umn.edu
21

368
­therapy and has been recently become widely uti-
lized for the management of androgenetic alope-
cia (AGA) [1, 2]. The initial discovery was made
by Dr. Endre Mester in 1967 when he serendipi-
tously discovered that mice irradiated with low-­
powered lasers experienced increased hair
regrowth [3, 4]. Since that time, the market for
PBM devices for the management of alopecia has
rapidly expanded.
Many of the available designed PBM devices
have been focused on AGA, as it is the most com-
mon of the alopecias in both sexes [5–8]. AGA
not only results in physical changes and hair thin-
ning of the scalp, it has also been associated with
psychological issues such as reduced quality of
life and poor body image, making the develop-
ment of treatment options imperative [9–11].
Treatments for AGA include finasteride, topical
minoxidil, and for women, spironolactone may
be recommended. These treatments can be effec-
tive, however, they may be associated with local
or systemic side effects. Hair transplantation may
be offered, especially for extensive disease, how-
ever, this is often costly and involves downtime.
More recently, platelet rich plasma (PRP) therapy
has been proposed as a treatment option, though
large randomized controlled trials supporting use
are lacking. To date, none of these devices have
been Food and Drug Administration (FDA)
cleared for the management of other non-­
cicatricial or cicatricial alopecias.
Mechanism
The exact mechanism of PBM therapy is
unknown and remains to be elucidated.
However, preliminary work has found PBM
encourages tissue regeneration, alleviates pain,
and decreases inflammation, making it applica-
ble to medical fields ranging from dentistry and
pain to acupuncture and dermatology [2, 12–
19]. At the cellular level, it has been proposed
that PBM changes adenosine triphosphate pro-
duction in the mitochondria [2, 20, 21]. The
anti-inflammatory properties are postulated to
decrease pro-­inflammatory cytokines [2, 13,
22]. PBM is also thought to effect transcription
factor induction and cell-signaling, therefore
modifying gene expression along with cell
migration, proliferation and metabolism [2, 21,
23–28]. With regards to its use for hair growth,
it has been hypothesized to stimulate bulge
stem cells within the hair follicle and aid in the
transition of the follicles from telogen to ana-
gen [2, 29]. It is also thought to extend anagen,
inhibit early transition to catagen, and acceler-
ate hair growth [2, 29].
Photobiomodulation Device
Characteristics
PBM devices may contain exclusively diode
lasers or lasers combined with light-emitting
diodes (LEDs). Lasers emit coherent, monochro-
matic light, whereas LEDs emit non-coherent
light with a wider range of wavelengths [2]. The
continuous laser light has previously been
thought to be imperative for PBM efficacy.
However, the use of LEDs has challenged this
theory, and whether diode lasers or LEDs are
more clinically efficacious remains to be deter-
mined [2]. Wavelengths of these devices range
from 500 to 1000 nanometers (nm) to encom-
pass the red or near-infrared spectrum of light
[2]. Amongst the PBM devices currently avail-
able on the market, the total number of lasers
and/or LEDs ranges from 7 to 272 diodes [30–
45]. The total power for each device ranges
between  approximately 35 milliwatts (mW) to
1360  mW.  The iGrow® and iRestore® are the
only devices currently incorporating both laser
diodes and LEDs into their design. Marketed
devices may emit light in a continuous or pulsed
fashion. Of note, the most efficacious wave-
length, power, treatment time and frequency for
these devices to stimulate hair growth remains to
be elucidated.
In 2007, the FDA cleared the first PBM device
for the treatment of AGA [46]. The affordability,
non-invasive nature, and convenient at home or
office-based treatment options have sparked
patient interest in use of the PBM devices when
compared to procedures such as hair transplanta-
tion. According to the FDA 510(k) Premarket
M. B. Hirt and R. S. Farah

369
Notification database, there are currently 16
PBM devices marketed for home-use in the
United States (Table 21.1) [47]. The cost of each
device ranges from approximately $295 to $3000,
often based on the number of diodes and shape of
the device [30–45]. Available device shapes
include combs, helmets, caps, or headbands.
Treatment time also varies based on the chosen
device and ranges from 90 seconds three time per
week to 36 minutes every other day. Response to
use of these PBM devices may take 3–4 months
of continued use, and the response is expected to
continue throughout the first 6–12 months of use
[30–45, 48]. The longest duration of treatment
reported in the literature is 26  weeks [48–50].
LaserCap® is currently the only device company
which limits sales to authorized physicians. All
other device companies offer their devices
directly to consumers.
PBM in the Home and Office
Consumers may purchase most of the available
PBM devices for home use. Two devices that
have been available for office-based treatments
are the Capillus272 Office Pro® and the Sunetics
Clinical Laser® [30, 31]. Office-based devices are
desirable for patients that would like to trial the
device prior to purchasing for home use.
Anecdotally, physicians have reported using
PBM after PRP in the office setting. However,
long-term studies on the efficacy of this tech-
nique are lacking. More recently, PBM device
sales have been expanded from the consumers
and physicians to hair salons. The remaining
devices on the market are designed for home use
(Table 21.1).
Available Data
As PBM continues to emerge as a treatment for
AGA, research within the field continues to
expand. In 2016, Zarei et al. reviewed 21 studies
(2 in vitro, 7 animal, and 12 clinical) to evaluate
the effectiveness of PBM in treating various types
of alopecia [50]. In all but two small, uncon-
trolled trials, PBM was found to significantly
increase hair regrowth [50]. In 2017, Afifi et al.
completed a systematic review of 11 studies, 5 of
which were randomized sham-controlled trials
[51]. Hair counts or density were used as objec-
tive measurements. Other evaluated items in sev-
eral studies included the following: patient
satisfaction, hair shaft diameter, tensile strength
and hair thickness. Hair thickness and tensile
strength improved in two of four studies evalu-
ated [51].
Published randomized controlled trials (RCT)
exist for HairMax®, iGrow®, and Capillus® PBM
devices. HairMax® LaserComb has conducted
the largest studies evaluating the effectiveness of
PBM devices in treating AGA. In 2009, the first
multicenter, double-blind RCT evaluated the
HairMax® LaserComb as compared to a sham
device in males with AGA [49]. Subjects assigned
to the HairMax® LaserComb in a 26-week study
period demonstrated a statistically significant
increase in mean hair density when compared to
controls. Subsequently in 2014, Jimenez et  al.
published a multicenter, double-blind RCT com-
paring the effectiveness of the HairMax®
LaserComb to a sham device in the treatment of
AGA including both men and women [48].
Subjects randomized to the HairMax® LaserComb
device achieved a statistically significant increase
in density of terminal hair fibers when compared
to subjects treated with the sham device.
In 2013, Lanzafame et al. evaluated the effi-
cacy of the iGrow®, a device with a combination
of lasers and LEDs, in male subjects with AGA
and reported a 35% increase in post-treatment
hair counts compared to controls using a sham-­
device [52]. Subsequently in 2014, Lanzafame
et al. reported a 37% increase in post-treatment
hair counts in female subjects using the iGrow®
device [53]. The most recently published study
was conducted by Friedman et al. in 2017. In this
study female AGA patients were randomized to
the Capillus® 272 device or a sham-device treat-
ment. Subjects used the devices every other day
for a total of 17  weeks. Subjects assigned the
Capillus® 272 device demonstrated a 51%
increase in hair counts as compared to the sham
device [54].
21  Photobiomodulation and Hair Growth

370
Table 21.1  Summary of clinical studies evaluating device efficacy for treatment of androgenetic alopecia
Studies Study type
Device
Subjects Treatment
Results
Peer-
reviewed
Capillus
Yes
(1)
Double blind,
sham
device-
controlled
multicenter
RCT
Handi-Dome
Laser (272
diode model)
44 F
30 min
Eevery other
day for
17 weeks
Statistically
significant 51%
increase in
terminal hair count
versus sham-
treated group
Yes
Friedman
et al. [54]
HairMax
Yes
(5)
Prospective
cohort study
HairMax
LaserComb
28 M
7 F
5–10 min
every other
day for
24 weeks
Total hair counts
increased by
93.5% and total
hair tensile
strength increased
by 78.9%
Yes
Satino
et al. [57]
Double blind,
sham
device-
controlled,
multicenter
RCT
HairMax
LaserComb
110 M
15 min
3 days/week
for 26 weeks
Mean terminal hair
density increased
by 19.8 hairs/cm2
versus 7.6 hairs/
cm2 decrease in
sham-treated
group
Device-treated
patients reported
overall
improvement in
hair health and
quality versus
sham-treated
group
Investigator’s
subjective global
assessment of hair
growth was not
significantly
different between
groups
Yes
Leavitt
et al. [49]
Case report
HairMax
LaserComb
2 M
7.5 min
3 days/week
for 26 weeks
No significant
change in hair
counts (total,
vellus, anagen,
telogen) or hair
thickness
Yes
Rushton
et al. [58]
Retrospective
cohort study
HairMax
LaserComb (7,
9 or 12 diode
model)
11 M
21 F
8, 11 or
15 min
3 days/
weekfor
8–48 weeks
Global
photographic
assessment of hair
growth showed 8
patients with
significant
improvement, 20
with moderate
improvement and
4 with no
improvement
Yes
Munck
et al. [59]
M. B. Hirt and R. S. Farah

371
Double blind,
sham
device-
controlled,
multicenter
RCT
HairMax
LaserComb (7,
9 or 12 diode
model)
128 M
141 F
8, 11 or
15 min
3 days/week
for 16 weeks
Overall, terminal
hair density
increased by 15.27
hairs/cm2
compared to
sham-treated
group
Higher percentage
of device-treated
patients reported
improvement in
hair loss, but this
did not always
reach statistical
significance
Yes
Jimenez
et al. [44,
48]
iGrow
Yes
(2)
Double blind,
sham
device-
controlled
RCT
TOPHAT655
(iGrow)
41 M
25 min
every other
day for
16 weeks
Statistically
significant 35%
increase of in
terminal hair count
versus sham-
treated group
Yes
Lanzafame
et al. [52]
Double blind,
sham
device-
controlled
RCT
TOPHAT655
(iGrow)
42 F
25 min
every other
day for
16 weeks
Statistically
significant 37%
increase of in
terminal hair count
versus sham-
treated group
Yes
Lanzafame
et al. [53]
iRestore
Yes
(1)
Double blind,
sham
device-
controlled
RCT
iRestore
18 M
18 F
25 min
3 days/week
for 17 weeks
Pending, study in
progress
N/A
LaserCap
Yes
(2)
Case series
LaserCap
7 F
30–60 min
3–4 days/
week
for
3–6 months
Improvement in
hair volume and
shine
No
Case series
LaserCap
1 M
2 F
30 min
every other
day for
6 months
Improvement in
hair volume and
shine
No
NutraStim
No
N/A
N/A
N/A
N/A
N/A
N/A
Theradome
Yes
(1)
Double blind,
sham
device-
controlled
multicenter
RCT
Theradome™
LH80 PRO
80 M
20 min
2 days/week
for 26 weeks
Pending, study in
progress
N/A
RCT randomized-controlled trial, M male, F female, N/A not applicable
Table 21.1  (continued)
In May 2017, Esmat et al. published a study
comparing the efficacy of the iGrow® device to
topical minoxidil 5% as well as the combination
of both therapies in treatment for female pattern
hair loss [55]. The outcomes were measured
using folliscope and ultrasound bio-macroscopic
(UBM) techniques. This preliminary investiga-
tion suggested that use of a combination PBM
21  Photobiomodulation and Hair Growth

372
with minoxidil could accelerate hair density
when compared to either modality used alone.
Limitations of this study include the use of a fol-
liscope and UBM techniques for evaluation as
these have not been used frequently for this type
of assessment in the past [55].
Safety Profile
One of the benefits of the PBM devices for the
treatment of AGA is their outstanding safety pro-
file. The most common side effects reported in the
large multicenter study involving 269 subjects
were pruritus (2.5%) and dry skin (5.1%) [48].
Other less common side effects of using these
devices are irritation, scalp tenderness, mild urti-
caria, redness, acne, and a warm sensation [48].
The primary concern with use of laser devices is
the risk of retinal damage as a result of laser light
exposure [56]. To the authors’ knowledge, no
reports of ocular damage from PBM devices are
reported. As a means to further prevent this risk,
many of the devices currently on the market con-
tain a built-in safety feature to help prevent acci-
dental eye exposure. Data on use in pregnant and
lactating patients are lacking.
Clinical Pearls
Clinical experiences with these devices may lead
dermatologists to find that cost is a barrier for
their use. In these situations, combs are the least
expensive and may be considered. Other issues
encountered in clinic are varying hair thickness,
texture and curl. Some clinicians may lean
towards using a device with teeth so as to part
thick hair and possibly deliver more light to the
target. Anecdotally, compliance is also an issue,
even with extensive counseling from a seasoned
clinician. In these situations one may consider a
cap or helmet shaped device which is hands free.
Patients with extensive hair loss may also find
hands free devices helpful. There are dermatolo-
gists in the United States using these devices off-­
label for the treatment of other cicatricial and
noncicatricial alopecias. Data for use in other
types of hair loss is lacking.
Conclusion and Future Directions
Evidence suggests that PBM is a safe and effec-
tive
treatment
option
for
patients
with
AGA. Therefore, it may be an effective alterna-
tive treatment for patients who are unresponsive
to medical management or do not wish to undergo
surgical procedures or take medications to treat
their disease. Further studies are needed to exam-
ine the effectiveness of PBM devices in the treat-
ment of other types of alopecia (i.e. telogen
effluvium, lichen planopilaris, frontal fibrosing
alopecia, etc.) and varying types including thick,
thin, curly and straight hair. Research focused on
the development of topical and oral treatment
protocols in conjunction with PBM should con-
tinue to be pursued [55].
There are numerous devices on the market,
making it difficult for the physician and patient to
navigate. Currently, it is unknown if one device
offers more clinical benefit over another. A head-­
to-­head comparison of FDA-cleared PBM
devices was initiated in 2016 and results have not
yet been reported. Each device has unique design
features. In addition, these devices vary in terms
of cost, total power output, frequency of use, and
treatment time. Wavelengths of these devices are
similar, though optimal treatment wavelength is
unknown. Further research into device specifica-
tions such wavelength, dosimetry, frequency of
use, duration of treatment, and optimal light
source is needed to determine the most effective
treatment modality for patients with AGA. Each
of these factors is important for patients when
considering a PBM device. Physicians should
understand PBM and remain informed and armed
with information regarding devices on the mar-
ket. As this arena continues to expand, questions
regarding PBM will likely become more com-
monplace in the office setting.
References
1. Anders JJ, Lanzafame RJ, Arany PR. Low-level light/
laser therapy versus photobiomodulation therapy.
Photomed Laser Surg. 2015;33(4):183–4.
2. Chung H, Dai T, Sharma SK, Huang YY, Carroll JD,
Hamblin MR. The nuts and bolts of low-level laser
(light) therapy. Ann Biomed Eng. 2012;40(2):516–33.
M. B. Hirt and R. S. Farah

373
3. Mester E, Szende B, Tota JG. Effect of laser on hair
growth in mice. Kiserl Orvostud. 1967;19:628–31.
4. Mester E, Szende B, Gartner P.  The effect of laser
beams on the growth of hair in mice. Radiobiol
Radiother (Berl). 1968;9(5):621–6.
5. Severi G, Sinclair R, Hopper JL, English DR,
McCredie MR, Boyle P, Giles GG. Androgenetic alo-
pecia in men aged 40-69 years: prevalence and risk
factors. Br J Dermatol. 2003;149(6):1207–13.
6. Gan DC, Sinclair RD. Prevalence of male and female
pattern hair loss in Maryborough. J Investig Dermatol
Symp Proc. 2005;10(3):184–9.
7. Hamilton JB. Patterned loss of hair in man: types and
incidence. Ann N Y Acad Sci. 1951;53(3):708–28.
8. Norwood OT.  Incidence of female androgenetic
alopecia (female pattern alopecia). Dermatol Surg.
2001;27(1):53–4.
9. Han SH, Byun JW, Lee WS, Kang H, Kye YC, Kim
KH, Kim DW, Kim MB, Kim SJ, Kim HO, Sim
WY, Yoon TY, Huh CH, Hwang SS, Ro BI, Choi
GS. Quality of life assessment in male patients with
androgenetic alopecia: result of a prospective, multi-
center study. Ann Dermatol. 2012;24(3):311–8.
10. Cash TF.  The psychological effects of andro-
genetic alopecia in men. J Am Acad Dermatol.
1992;26(6):926–31.
11. Cash TF, Price VH, Savin RC. Psychological effects
of androgenetic alopecia on women: comparisons
with balding men and with female control subjects. J
Am Acad Dermatol. 1993;29(4):568–75.
12. Kovács IB, Mester E, Görög P. Stimulation of wound
healing with laser beam in the rat. Experientia.
1974;30(11):1275–6.
13. Lim W, Choi H, Kim J, Kim S, Jeon S, Zheng
H, Kim D, Ko Y, Kim D, Sohn H, Kim O.  Anti-­
inflammatory effect of 635 nm irradiations on in vitro
direct/indirect irradiation model. J Oral Pathol Med.
2015;44(2):94–102.
14. Chow RT, Johnson MI, Lopes-Martins RA, Bjordal
JM. Efficacy of low-level laser therapy in the man-
agement of neck pain: a systematic review and meta-­
analysis of randomised placebo or active-treatment
controlled trials. Lancet. 2009;374(9705):1897–908.
15. Gigo-Benato D, Geuna S, Rochkind S. Phototherapy
for enhancing peripheral nerve repair: a review of the
literature. Muscle Nerve. 2005;31(6):694–701.
16. Hashmi JT, Huang YY, Osmani BZ, Sharma SK,
Naeser MA, Hamblin MR.  Role of low-level laser
therapy in neurorehabilitation. PM R. 2010;2(12
Suppl):S292–305.
17. Bjordal JM, Johnson MI, Iversen V, Aimbire F,
Lopes-­Martins RA.  Low-level laser therapy in
acute pain: a systematic review of possible mecha-
nisms of action and clinical effects in randomized
placebo-controlled trials. Photomed Laser Surg.
2006;24(2):158–68.
18. Pandeshwar P, Roa MD, Das R, Shastry SP, Kaul R,
Srinivasreddy MB. Photobiomodulation in oral medi-
cine: a review. J Investig Clin Dent. 2016;7(2):114–26.
19. Avci P, Gupta A, Sadasivam M, Vecchio D, Pam Z,
Pam N, Hamblin MR. Low-level laser (light) therapy
(LLLT) in skin: stimulating, healing, restoring. Semin
Cutan Med Surg. 2013;32(1):41–52.
20. Greco M, Guida G, Perlino E, Marra E, Quagliariello
E.  Increase in RNA and protein synthesis by mito-
chondria irradiated with helium-neon lase. Biochem
Biophys Res Commun. 1989;163:1428–34. [PubMed:
2476986]
21. Karu TI. Primary and secondary mechanisms of action
of visible to near-IR radiation on cells. J Photochem
Photobiol B. 1999;49:1–17. [PubMed: 10365442]
22. Fukuda TY, Tanji MM, Silva SR, Sato MN, Plapler
H.  Infrared low-level diode laser on inflam-
matory process modulation in mice: pro- and
anti-­inflammatory cytokines. Lasers Med Sci.
2013;28(5):1305–13.
23. Avci P, Gupta GK, Clark J, et  al. Low-level laser
(light) therapy for treatment of hair loss. Lasers Surg
Med. 2014;46(2):144–51.
24. Farivar S, Malekshahabi T, Shiari R.  Biological
effects of low level laser therapy. J Lasers Med Sci.
2014;5(2):58–62.
25. De Marchi T, Leal Junior EC, Bortoli C, Tomazoni
SS, Lopes-Martins RA, Salvador M.  Low-level
laser therapy (LLLT) in human progressive-intensity
running: effects on exercise performance, skeletal
muscle status, and oxidative stress. Lasers Med Sci.
2012;27(1):231–6.
26. Moriyama Y, Nguyen J, Akens M, Moriyama EH,
Lilge L. In vivo effects of low level laser therapy on
inducible nitric oxide synthase. Lasers Surg Med.
2009;41(3):227–31.
27. Huang YY, Nagata K, Tedford CE, McCarthy T,
Hamblin MR.  Low-level laser therapy (LLLT)
reduces oxidative stress in primary cortical neurons
in vitro. J Biophotonics. 2013;6(10):829–38.
28. Zhang Y, Song S, Fong CC, Tsang CH, Yang Z, Yang
M. cDNA microarray analysis of gene expression pro-
files in human fibroblast cells irradiated with red light.
J Invest Dermatol. 2003;120(5):849–57.
29. Wikramanayake TC, Rodriguez R, Choudhary S,
et  al. Effects of the Lexington lasercomb on hair
regrowth in the C3H/HeJ mouse model of alopecia
areata. Lasers Med Sci. 2012;27(2):431–6.
30. Capillus272 Pro, Capillus272 OfficePro, Capillus82,
Capillus202 510(k) Summary. Available from: https://

pdf. Accessed 1 Dec 2016.
31. Sunetics International. Sunetics Lasers. Available
from: Accessed 5 Dec
2016.
32. Capillus LLC.  Available from:
Accessed 1 Dec 2016.
33. HairMax® LaserComb 510k Premarket Application.
Available
from:

cdrh_docs/pdf10/K103368.pdf. Accessed 1 Dec
2016.
34. Lexington International, LLC.  HairMax. Available
from: Accessed 1 Dec 2016.
35. igrow-II Hair Growth System 510(k) Summary.
Available from:
docs/pdf14/k141567.pdf. Accessed 1 Dec 2016.
21  Photobiomodulation and Hair Growth

374
36. Apira Science, Inc. iGrow® Hair Growth System.
Available from: Accessed 1
Dec 2016.
37. iRestore Hair Growth System (Model ID-500) 510(k)
Summary. Available at:
gov/cdrh_docs/pdf15/K151662.pdf. Accessed 1 Dec
2016.
38. Freedom Laser Therapy, Inc. iRestore Hair Growth
System.
Available
at:

Accessed 1 Dec 2016.
39. Lasercap 510(k) Summary. Available from: http://
pdf16/K161875.
pdf. Accessed 1 Dec 2016.
40. Transdermal Cap, Inc. LaserCap. Available at: www.
lasercap.com. Accessed 1 Dec 2016.
41. Nutra Stim Hair Laser Comb Model NSL-6318 510(k)
Summary. Available from:
fda.gov/cdrh_docs/pdf14/K141588.pdf. Accessed 1
Dec 2016.
42. NutraStim, LLC. Available from:
com. Accessed 1 Dec 2016.
43. Theradome Laser Helmet LH80 PRO 510(k)
Summary. Available from:
fda.gov/cdrh_docs/pdf12/K122950.pdf. Accessed 1
Dec 2016.
44. Theradome, Inc. Available from:
com. Accessed 1 Dec 2016.
45. EGLOBAL, LLC. Illumiflow. Available from: https://
illumiflow.net. Accessed 5 April 2017.
46. FDA Hairmax lasercomb. Available online at: http://

CDRH/CDRHFOIAElectronicReadingRoom/
UCM203056.pdf
47. 510(k) Premarket Notificaton Database. U.S.  Food
and Drug Administration. Available at:
Accessdata.fda.gov/scripts/cdrh/cfdocs/cfPMN/pmn.
cfm. Accessed 1 Dec 2016.
48. Jimenez JJ, Wikramanayake TC, Bergfeld W,
Hordinsky M, Hickman JG, Hamblin MR, Schachner
LA. Efficacy and safety of low-level laser device in the
treatment of male and female pattern hair loss: a mul-
ticenter, randomized, sham device-controlled, double-­
blind study. Am J Clin Dermatol. 2014;15(2):115–27.
49. Leavitt M, Charles G, Heyman E, Michaels
D.  HairMax LaserComb laser phototherapy device
in the treatment of male androgenetic alopecia: a ran-
domized, double-blind, sham device-controlled, mul-
ticenter trial. Clin Drug Investig. 2009;29(5):283–92.
50. Zarei M, Wikramanayake TC, Falto-Aizourua L,
Schachner LA, Jimenez JJ. Low level laser therapy
and hair regrowth: an evidence-based review. Lasers
Med Sci. 2016;31(2):363–71.
51. Afifi L, Maranda EL, Zarei M, Delcanto GM, Falto-­
Aizpurua L, Kluijfhout WP, Jimenez JJ.  Low-level
laser therapy as a treatment for androgenetic alopecia.
Lasers Surg Med. 2016;49(1):27–39.
52. Lanzafame RJ, Blanch RR, Bodian AB, Chiacchierini
RP, Fernandez-Obregon A, Kazmirek ER. The growth
of human scalp hair mediated by visible red light
laser and LED sources in males. Lasers Surg Med.
2013;45(8):487–95.
53. Lanzafame RJ, Blanche RR, Chiacchierini RP,
Kazmirek ER, Sklar JA. The growth of human scalp
hair in females using visible red light laser and LED
sources. Lasers Surg Med. 2014;46(8):601–7.
54. Friedman S, Schnoor P.  Novel approach to treat-
ing androgenetic alopecia in females with photo-
biomodulation (low-level laser therapy). Dermatol
Surg.
2017;43:856.

DSS.0000000000001114.
55. Esmat S, Hegazy R, Gawdat H, et al. Low-level light
minoxidil 5% combination versus either therapeutic
modality alone in management of female patterned
hair loss: a randomized controlled study. Lasers Surg
Med. 2017;49:835.
56. Schulmeister K, Jean M.  The risk of retinal injury
from Class 2 and visible Class 3R lasers, includ-
ing medical laser aiming beams. Med Laser Appl.
2010;25:99–110.
57. Satino JL, Markou M. Hair regrowth and increased
hair tensile strength using the HairMax LaserComb
for low-level laser therapy. Int J Cosmetic Surg
Aesthetic Dermatol. 2003;5(2):113–7.
58. Rushton DH, Gilkes JJH, Van Neste DJJ.  No
improvement in male-pattern hair loss using laser
hair-comb therapy: a 6-month, half-head, assessor-­
blinded investigation in two men. Clin Exp Dermatol.
2012;37(3):313–5.
59. Munck A, Gavazzoni  MF,  Trüeb RM.  Use of low-­
level laser therapy as monotherapy or concomitant
therapy for male and female androgenetic alopecia.
Int J Trichology. 2014;6(2):45–9.
M. B. Hirt and R. S. Farah

375
© Springer International Publishing AG, part of Springer Nature 2018
K. Nouri (ed.), Lasers in Dermatology and Medicine,
Reflectance Confocal Microscopy
in Oncological Dermatology
Pablo Fernández-Crehuet Serrano,
Gonzalo Segurado-Miravalles,
and Salvador González
Abstract
Imaging techniques capable of non-invasive,
high-resolution, skin imaging in vivo have been
the focus of recent attention in the dermatology
field. These efforts are directed to improve the
diagnostic accuracy of skin cancer, especially
cutaneous melanoma. Reflectance-mode con-
focal microscopy (RCM) allows real time non-
invasive histological imaging with cytological
detail comparable to conventional histology of
the skin when exploring cutaneous structures
between the stratum corneum and the upper
reticular dermis. RCM consists of a light source
for illumination of a small spot within translu-
cent tissue; and a point detector that detects
back-­scattered and reflected light though a pin-
hole. The pinhole prevents out-of-focus light
from reaching the detector; as a result, only the
optical plane in focus (confocal) is detected.
Similar to dermoscopy images, real time
images obtained by RCM are oriented horizon-
tal to skin surface (optical transversal sections).
Melanin provides strong contrast because of its
high refractive index (1.7) relative to the sur-
rounding epidermis; the melanosome size,
­similar to the near-infrared wavelengths (800–
1064 nm), produces strong back scattering of
the beam. Thus, cells containing melanin, such
as pigmented keratinocytes, melanocytes, or
melanophages, appear very bright when illumi-
nated in this manner. Similarly, other organelles
or cytoplasmic granules provide good contrast
(albeit less intense than melanin), resulting in
good imaging of cells containing them, such as
Langerhans cells, lymphocytes, or cytoplasmic
granules in keratinocytes at the stratum granu-
losum. Major confocal imaging criteria of pig-
mented lesions such as benign and malignant
melanocytic tumors have been established.
RCM imaging criteria of other skin cancers
such as basal cell carcinoma, squamous cell
carcinoma, oral cavity neoplasm, and mycosis
fungoides have also been evaluated. RCM
shows promise for: (1) guidance during biopsy
collection; (2) monitoring histological architec-
tural changes or dynamic process such as
inflammatory response or capillary flow; (3)
histological correlation with dermoscopic fea-
tures; (4) monitoring of the response of a given
lesion to treatment; and (5) demarcation of the
P. F.-C. Serrano
Department of Dermatology,
Alto Guadalquivir Hospital, Andújar, Jaén, Spain
G. Segurado-Miravalles
Department of Dermatology, Ramón y Cajal Hospital,
Madrid, Spain
S. González (*)
Department of Medicine and Medical Specialities,
Alcalá University, Madrid, Spain
Dermatology Service, Memorial Sloan-Kettering
Cancer Center, New York, NY, USA
Hospital Ramón y Cajal, Alcala University,
Madrid, Spain
e-mail: gonzals6@mskcc.org
22

376
extension of a lesion before proceeding to inva-
sive treatments such as surgical excisions. The
main limitation of RCM is its relatively low
penetration through the dermis; currently, a
maximum depth of 250–300  μm can be
achieved, preventing imaging of structures
located in deep dermis and hypodermis. The
main challenge is the interpretation of images.
Specific photographic atlas, courses and devel-
opment of teledermatology may solve this
problem.
Keywords
Confocal microscopy · Melanoma · Nevi
Basal cell carcinoma · Actinic keratoses
Squamous cell carcinoma · Bowen disease
Introduction
Dermatology is a medical speciality in which
diagnosis is frequently based exclusively on
clinical examination. Recent advances in imag-
ing techniques provide the potential for non-
invasive high-resolution skin imaging in  vivo.
These can overcome some of the disadvantages
of the conventional biopsy and histologic analy-
sis. Such advances include dermoscopy [1],
optical coherence tomography [2], high-fre-
quency ultrasound [3], magnetic resonance
imaging
[4],
fluorescence-­mode
confocal
microscopy [5], and reflectance-mode confocal
microscopy (RCM). Of these, confocal micros-
copy is the technique with more possible appli-
cations,
offering
real
time
non-invasive
microscopic images with the highest resolution
imaging comparable to routine histology when
exploring cutaneous structures between stratum
corneum and reticular dermis [6–8].
Principles of Reflectance-Mode
Confocal Microscopy
The confocal scanning microscope was invented
by Marvin Minsky while working as postdoctoral
fellow at Harvard in 1955 [9]. However, it
required the development of light source and
computerization technologies to enable realiza-
tion of tissue imaging in vivo. Since the 1980s
several research groups have demonstrated the
use of tandem scanning confocal microscopy for
imaging human and animal tissue in vivo [10–
13]. Confocal scanning laser microscopy for
imaging human skin in vivo was first reported in
1995 [8]. In vivo RCM offers several important
advantages over conventional histology. Imaging
is painless and non-invasive, causing no tissue
damage. The skin is not altered in any by pro-
cessing (fixation, sectioning, and mounting) or
staining, minimizing artifact or disruption of the
native structure in the tissue. The data, collected
in real time, is faster than routine histology, and
the skin site can be repeatedly imaged over time
to evaluate dynamic changes such as tissue
growth, wound healing, lesion progression or
response to therapy [14–18]. Conventional his-
tology only demonstrates tissue morphology at
one time point.
The principle of reflectance confocal micros-
copy involves the use of a point source of light
that illuminates a small spot within translucent
tissue. The reflected light (reflectance) is then
imaged onto a detector after passing through a
small pinhole. The pinhole prevents out-of-focus
light from reaching the detector. This means that
only the region of a specimen that is in focus
(confocal) is detected. To create an image of the
whole plane of the sample being studied, the
point source beam is scanned. This enables a vir-
tual sectioning of a thin horizontal tissue plane
in vivo. The ability to optically section a tissue
enables intact tissue to be imaged without the
need to physically section the tissue as is the case
with conventional histology, allowing the physi-
cal structure of the tissue to be preserved. The
resolution provided by RCM depends on the pin-
hole size, the numerical aperture of the objective
lens, and the wavelength used. Lasers of different
wavelengths may be used as the light source for
RCM. Longer wavelengths penetrate deeper into
the skin but provide less lateral resolution. Back-­
scattering of light occurs due to local variations
of the refractive index within the tissue as well as
when the scattering structure has a size similar to
P. F.-C. Serrano et al.

377
the illuminating wavelength. Near-infrared wave-
lengths (800–1064  nm) produce strong back-­
scattering from melanosomes, despite melanin
absorption at this wavelength, because they have
a high refractive index relative to the surrounding
epidermis and have a size similar to the illumi-
nating wavelength [14]. This means that cells
containing melanin, such as basal keratinocytes
and melanocytes, image brightly.
New microscopes use a single optical fibre that
is common to both the illumination optical path
and the detection optical path. This single-­mode
optical fibre acts as a spatial filter which rejects
the out-of-focus information collected by the
objective lens. This system simplifies the opto-
mechanical complexity by eliminating the bulky
optical components and confocal apertures (pin-
holes) associated with conventional confocal
implementations. This fibre-optic approach has
enabled miniaturization of the confocal scanner
into a hand-held device that provides the flexibil-
ity and mobility necessary for current clinical use.
The commercially available RCM has a wave-
length of 830 nm and 30× objective lens of NA
0.9, which provides a lateral resolution of approx-
imately 1  μm and an axial resolution (section
thickness) of 3–5 μm [14]. With this system, it is
possible to image normal skin to a depth of 200–
250 μm [14]. This is sufficient for imaging epi-
dermis and upper dermis (papillary dermis and
upper reticular dermis). Imaging deeper layers
within the skin can also be achieved by using
greater laser power, but the laser power used in
the commercially available device is less than
30  mW and causes no tissue damage or eye
injury. Water immersion lenses are used since the
refractive index of water (1.33) is close to that of
epidermis (1.34) and this minimizes spherical
aberrations caused when the light passes through
the tissue-air interface [14]. It is also possible to
use water-based gels as immersion media, par-
ticularly if imaging a scaly or hyperkeratotic
lesion since the gel settles between disrupted cor-
neocytes, reducing irregularities in refraction.
Gel is also useful if imaging a skin site that is not
particularly flat, since the gel doesn’t run off of
the skin in the same way water can. A skin con-
tact device is used to reduce motion artefact and
contain the water or the gel interface when imag-
ing. This device consists of a metal ring that is
fixed to the patient’s skin with adhesive, and is
coupled to the microscope housing with a magnet
during imaging. It has a concave shape to hold
the immersion medium. By moving the objective
lens in the “z” stage (vertical) with respect to the
skin surface, it is possible to image at different
horizontal levels within the tissue since the focal
plane is progressively moved deeper. Images can
be grabbed to produce static pictures of horizon-
tal skin sections as well as recorded on videotape
(20–30  Hz) to produce movies to demonstrate
dynamics events such as blood flow [8, 14–18].
Reflectance Confocal Microscopy
Findings of Normal Skin
Confocal microscopy offers a new view of the skin,
both in terms of orientation and image content,
with two principal differences from routine histol-
ogy. First, the image obtained is horizontal or en
face (horizontal) rather than vertical sections that
are normally obtained from routine histology.
Second is that this image is in a gray-scale (bright-
scale) similar to radiographs. The field of view with
RCM varies with different microscopes, but is gen-
erally 250–500  μm across [14]. The level being
imaged can be ascertained by the morphologic
appearance of tissue at a given depth or by measur-
ing the depth of section, using a micrometer
attached to the “z” stage of the objective lens.
Image contrast is produced by differences in the
refractive indices of the varying tissue and cell
structures. Melanin-containing structures (melano-
somes, melanocytes, melanophages, and pig-
mented keratinocytes, among others) have the
highest refractivity, followed by keratin-containing
structures, such as the stratum corneum, the infun-
dibulum, and the hair follicle. Nuclei, air, and
serum
exhibit
minimum
reflectivity
[19].
Microscopic structures similar in size to the wave-
length of the incident light display the highest
refractive indices. The maximum imaging depth
that has been achieved to date with RCM is approx-
imately 300 μm [14] although this may increase as
the technique continues its development.
22  Reflectance Confocal Microscopy in Oncological Dermatology

378
When imaging the skin in real time starting
from the surface and progressing deeper, the
most superficial images obtained are of the stra-
tum corneum. This produces very bright images
because the refractive difference at the interface
between the immersion medium (water at 1.33)
and stratum corneum (1.54) results in a large
amount of back-scattered light. Low laser fluen-
cies help to minimize this. The morphologic
appearance is that anucleated polygonal corneo-
cytes measuring 30–40 μm in size, and grouped
in “islands” separated by skin folds, which appear
very dark. The next layer seen is the stratum
granulosum consisting of 2–4 layers of cells
measuring 25–35 μm in size with a thickness of
between 3 and 10 cells, depending on the ana-
tomic location. These cells have the nuclei as
dark central ovals within the cell, surrounded by
bright grainy cytoplasm due to the presence of
bright multiple structures (0.1–1.0 μm) that cor-
respond to keratohyalin granules. The granulo-
cytes have clear outlines that form the
characteristic confocal finding known as the hon-
eycomb pattern. The spinous layer has between 5
to 10 layers of 15–25 μm in size cells, which have
a dark oval central area (the nucleus) and clearly
demarcated outlines that together with the stra-
tum granulosum form the characteristic honey-
comb pattern. The deepest layer of epidermis, the
basal layer, is seen as a bright clusters of cells
measuring about 7–10 μm [20]. The suprapapil-
lary epidermal plate at the dermo-epidermal
junction is apparent as rings of bright basal cells
surrounding a dark dermal papillae, which often
show a central area of blood flow consistent with
papillary dermal vascular loops. The papillary
dermis can be seen to consist of a network of
reticulated fibers and small blood vessels. We can
also observe eccrine ducts, with appear as bright
centrally hollow structures that spiral through
epidermis and dermis, and hair shafts with pilo-­
sebaceous units. These appear as whorled cen-
trally hollow structures with elliptical elongated
cells at the circumference and a central refractile
long hair shaft.
The appearance of normal skin varies accord-
ing to the skin site and skin colour being imaged
[21]. Skin from sun-exposed or darkly pigmented
sites appears generally brighter because of what
appears to be more pigment at the basal layer.
Sun-exposed skin also demonstrated thicker and
more fissured or wrinkled stratum corneum, more
randomly arranged and irregularly shaped dermal
papillae, and clumping of the dermal reticulated
pattern, consistent with collagen and elastic
fibers. Variation in the density of keratinocytes is
also apparent, with sun-protected sites showing a
greater density than sun-exposed sites. The palms
and soles of feet are notable for having an
extremely thick stratum corneum and a greater
number of eccrine ducts.
Reflectance Confocal Microscopy
of Non-melanocytic Neoplastic Skin
Lesions
RCM characterization of neoplastic lesions is an
important area for research, with the potential to
aid in the non-invasive diagnosis and manage-
ment of a variety of skin cancers. With the advent
of newer, less invasive, or topical therapies, it is
desirable to use a non-invasive diagnostic tool
that can allow high resolution, accurate identifi-
cation of tumor subtypes and tumor margins, and
response to treatment.
Basal Cell Carcinoma (Fig. 22.1)
Basal Cell Carcinomas (BCC) are the commonest
skin tumors in man, and RCM characteristics of
BCC have been well defined [22]. As with histol-
ogy, the different subtypes of BCC share certain
confocal patterns that make it relatively easy to
reach a diagnosis. They include the presence of
islands of monomorphic tumor cells (Fig. 22.1b)
that are elongated in shape and have nuclei ori-
ented along the same axis, producing a polarized
appearance (basaloid nuclei). This polarized cell
pattern persists through the thickness of the epi-
dermis, with loss of the normal progressive size
difference of differentiating epidermal cells, loss
of the normal honeycomb pattern, and loss of der-
mal papillae architecture. The presence of pleo-
morphism and architectural disorder of the
P. F.-C. Serrano et al.

379
overlying epidermis is indicative of actinic dam-
age or consequence of the tumor. In addition,
RCM images show numerous dark, round spaces
or branching (arborizing) lines, containing mov-
ing cells with different levels of refractivity. This
phenomenon corresponds to leukocyte trafficking
[23]. It is also possible to visualize inflammatory
cell infiltrate among the tumor cells. Imaging is
obtained at video-rate (30 frames per s), allowing
high temporal resolution (33  ms per frame) for
visualizing dynamic process such as leukocyte
rolling and adhesion on the endothelium. Another
relatively common finding is a dark area sur-
rounding aggregates of tumor cells, probably due
to mucin deposits. This feature corresponds to the
characteristic clefting (separation of tumor
islands from the surrounding stroma) seen on his-
tology (Fig.  22.1c). Ulrich et  al. presented 13
cases of BCC (including the three main histologic
subtypes) and found good linear correlation
between dark areas seen by RCM and peritumoral
mucin thickness [24]. The stroma sometimes
appears brighter than the tumor islands, which are
darker than usual (hyporeflective) (Fig. 22.1b, d)
and are referred to as dark silhouettes. This fea-
ture was first described by Ravinobitz and co-
workers and was described in detail [19]. Small
bright structures corresponding to inflammatory
cells can also be seen in the dermis.
All these observations allow defining a num-
ber of criteria to establish a successful diagnosis
of BCC using RCM. As the number of criteria is
higher, the specificity increased. A retrospective,
multicentric study from 152 lesions [25] has
shown that the presence of at least two of these
criteria has a sensitivity of 100% for the diagno-
sis of BCC. The presence of two or more criteria
was found to be 100% sensitive and 53.6% spe-
cific for the diagnosis of BCC, while four or more
RCM criteria presented 95.7% specificity and
82.9% of sensitivity.
In a published large prospective study, Guitera
et  al. [26] analyzed 710 consecutive equivocal
lesions by RCM. The lesions included 216 mela-
nomas, 266 nevi, 119 BCCs, 67 pigmented facial
macules, and 42 lesions classified as other skin
tumors. They studied 50% of the lesions (chosen
randomly) by multivariate analysis and identified
eight independently significant diagnostic features
for BCC, with a sensitivity of 97.1% and a speci-
250 mm
100 mm
150 mm
a
b
c
d
e
Fig. 22.1  Basal cell carcinoma. (a) Dermoscopy reveals
spoke-wheel areas and concentric blue-gray structures.
(b) RCM mosaic (1.5 × 1.5 mm) at the upper dermis level
revealing tumor nodules (asterisks) of weak to moderate
refractility. The bright stroma delineates individual tumor
islands. Red and blue rectangles correspond to magnified
images labeled c and d. (c) RCM view (0.250 × 0.5 mm)
shows a defined lobulated tumor island (asterisk) with
peripheral palisading of nuclei (yellow arrowheads),
subtle peritumoral dark cleft-like space, and a refractile
surrounding fibrotic stroma (s). (d) RCM view
(0.5 × 0.650 mm) shows a collection of tumoral islands
(asterisks) surrounded by brigt stroma (s). Small aggre-
gate of melanophages (red arrows) is also noted
22  Reflectance Confocal Microscopy in Oncological Dermatology

380
ficity of 93.4%. Five of the factors were positive:
polarized elongated structures in the superficial
layer, linear telangiectasia-like horizontal ves-
sels, compact nests of hyporeflective cells,
peripheral palisading, and a new concept called
epidermal shadowing, which they described as a
large dark featureless area disrupting the epider-
mis due to en face clefting of the underlying
tumor nests. The three negative features were the
atypical honeycomb pattern, nonvisible papillae,
and cerebriform nests. A recently published sys-
tematic review of diagnostic accuracy of RCM in
BCC estimated a sensitivity of 97% and a speci-
ficity of 93% of RCM in diagnosing BCC, this
supports outcomes of Guitera et al. [26, 27].
RCM has been employed in different studies
that describe the main features of different histo-
logical subtypes of BCC which is important in
order to determine the therapeutics and prognosis
of the tumor [28]. Infiltrative BCC is visualized
as ill-defined invading structures composed of
very polarized cells that penetrate and deform the
dermis, while nodular BCC reveals well-defined
tumor islands with peripheral palisading sur-
rounded by dark areas [29]. In this regard, Longo
et al. analyzed 88 BCC and correlated confocal
features and BCC subtype. Nodular BCC was
more likely if clefting was present and cords con-
nected to epidermis were absent. On the other
hand, infiltrative BCC was the most common
diagnosis if tumor islands, big or small, and cords
connected to epidermis were absent. Finally, the
presence of cords connected to epidermis in the
absence of clefting was associated with a higher
odds of superficial BCC [30].
Actinic Keratoses (Fig. 22.2)
Actinic keratoses (AK) are keratinocytic dyspla-
sias that can develop into squamous cell carcino-
mas (SCC). RCM features of AK include
irregular
hyperkeratosis,
and
parakeratosis
(Fig. 22.2b), architectural disarray (Fig. 22.2c),
epidermal cell nuclear enlargement with pleo-
morphism (Fig. 22.2c), and round vascular loops
frequently
surrounded
by
solar
elastosis
(Fig. 22.2d). The pattern of architectural disar-
ray does not involve the full thickness of the epi-
dermis [31]. Pellacani et  al. found good
concordance between the keratinocyte atypia
visualized with RCM and histopathology [32]. A
recently published study stated that photodam-
aged skin and AK are part of a disease contin-
uum, due to the almost constant presence of
keratinocyte pleomorphism and architectural
disruption in RCM images of photodamaged
skin, although less severe than in AK [33].
Ulrich and co-workers [34] evaluated the sen-
sitivity of RCM in the diagnosis of AKs. They
estimate this parameter in 97.7% (a total of 44
AKs were included in this study).
a
b
c
d
100 mm
100 mm
100 mm
se
se
Fig. 22.2  Actinic keratosis. Skin cancerization. (a)
RCM image (0.5 × 0.5 mm) at the level of stratum cor-
neum with hyperkeratinization and parakeratosis. (b)
RCM image (0.5 × 0.5 mm) at the spinous layer with kera-
tinocyte disarray (red dashed circle). (c) RCM image
(0.5 × 0.5 mm) of the upper dermis level displaying solar
elastosis (se) and round blood vessels (dashed red
circles)
P. F.-C. Serrano et al.

381
The most important limiting factor of RCM is
the shallow depth penetration of the illuminating
wavelength, which prevents accurate visualiza-
tion of the dermo-epidermal junction in particu-
larly hyperkeratotic lesions. This has traditionally
limited the capability of RCM to distinguish AK
from SCC.  Nevertheless new research in this
field conducted by Peppelman et  al. states that
the presence of architectural disarray in the stra-
tum spinosum and granulosum in combination
with dermal nest-like structures strongly suggests
a SCC instead of an AK [35].
Pigmented AKs usually constitute a diagnos-
tic challenge, in this way Moscarella et al. [36]
found the presence of epidermal changes (atypi-
cal keratinocytes, parakeratosis and scaling),
increased epidermal thickness, bright and small,
dermal papillae with enlarged interpapillary
space and intraepidermal dendritic cells as the
main RCM features of pigmented AK.
Bowen Disease
Bowen disease (BD) is an in situ squamous cell
carcinoma (SCC) histologically characterized by
proliferation of atypical pleomorphic keratino-
cytes throughout the epidermis. Dyskeratosis,
mitosis, and multinucleated cells are very
­common findings [37]. Clinically, it can be mis-
diagnosed as non-melanoma skin cancer or cer-
tain inflammatory skin conditions, such as
eczema and psoriasis [38]. Ulrich et  al. [39]
recently published a study of the confocal fea-
tures of ten BD lesions and described their cor-
relation with routine histologic features. The
most common confocal findings were disruption
of the stratum corneum, an atypical honeycomb
pattern in the epidermis with a greater degree of
architectural disorder and cellular atypia than in
AK, S-shaped blood vessels in the center of the
dermal papillae, and two types of characteristic
targetoid cells. The first type of cells was mor-
phologically large cells with a dark center, a
bright rim, and a dark halo, and the second type
were large cells with a bright center and a dark
halo. The cells are thought to correspond to the
different degrees of dyskeratosis seen by conven-
tional histology. Other features observed were
parakeratosis, multinucleated cells, and solar
elastosis.
Squamous Cell Carcinoma (Fig. 22.3)
In 2009, Rishpon et al. [40] published a study of
the confocal characteristics of 38 clinically sus-
pected SCC lesions that were subsequently con-
firmed by histology. The features identified were
an atypical honeycomb or disarranged pattern in
the epidermis (Fig. 22.3d), large round cells with
nuclear atypia in the stratum spinosum and stra-
tum granulosum (Fig.  22.3d), and round and
polymorphous blood vessels crossing the dermal
papillae (Fig. 22.3e). RCM images of the stratum
corneum typically reveal bright amorphous struc-
tures that correspond to the presence of crusts on
the tumor surface and polygonal nucleated cells
with a bright rim around a dark nucleus (para-
keratosis) (Fig. 22.3c). Hyperkeratosis and acan-
thosis permitting, RCM may also show increased
dermal vasculature and solar elastosis as well as
tumor islands in the case of invasive SCC [35].
As above mentioned, the presence of architec-
tural disarray in stratum spinosum and granulo-
sum in conjunction with nest-like structures in
dermis is associated with a correct diagnosis of
SCC in 88.5% of cases when discriminating
between SCC and AK [35].
Mycosis Fungoides
At the patch stage of mycosis fungoides (MF),
changes are often subtle; consequently diagnosis
at this early phase with confocal microscopy may
be difficult. The most important confocal features
include hyporefractivity of dermal papillary rings
and small, weakly refractive round cells located
in the spinous layer, which correlate to the inter-
face changes and exocytosis, respectively. In the
plaque phase of MF, the visualization of typical
confocal features of MF may be easier. Some
small, lightly refractive cells appear in the spi-
nous layer correlating to epidermotropism of
lymphocytes, which sometimes are grouped and
22  Reflectance Confocal Microscopy in Oncological Dermatology

382
located inside dark spaces within the epidermis
corresponding to Pautrier microabscesses on his-
topathology. These vesicle-like spaces have to be
distinguished from those seen in acute eczema by
the absence of parakeratosis and spongiosis and
the presence of other MF characteristic confocal
features. In tumor-type MF, the hyporefractive
papillary rings and the infiltration of small lightly
refractive cells in the epidermis are frequently
visualized in confocal images, while the vesicle-­
like spaces are rarely detected. Inside papillary
dermis, highly refractive cells of small to medium
size are observed and blood vessels may show a
well-circumscribed thickened wall [29]. RCM
may be a real-time guide for optimal biopsy site
selection in patients with multiple lesions sug-
gestive of MF.
Recently, a study published by Mancebo and
co-workers [41] showed a very good correlation
between RCM and histopathology regarding the
presence of epidermal lymphocytes and detection
of melanophages in the dermis. Pautrier collec-
tion had fair agreement (kappa 0.32), with Pautrier
collection more commonly visualized on RCM
(76% vs 59%). They also claimed that lesions
with Pautrier collection identified by RCM were
significantly more likely to show TCR clonality.
Pigmented Lesions
Early detection of melanoma is essential, and still
one of the most challenging problems in clinical
dermatology. The need for improved diagnostic
200 mm
150 mm
150 mm
150 mm
a
b
c
d
e
Fig. 22.3  Squamous Cell Carcinoma. (a) Dermoscopy
displays a nodular lesion with irregular and polymophous
vessels and whitish opaque areas. (b) Slight oblique RCM
submosaic (1.5 × 1.5 mm) at dermo epidermal junction
reveals keratinocyte disarray, exocytosis and polymor-
phous vessels (red arrowheads). In the upper portion
hyperkeratotic areas (hk) may be visualized. (c) RCM
image (0.5 × 0.5 mm) shows impetiginized stratum cor-
neum with inflammatory cells (yellow asterisks), atypical
nucleated corneocytes (red arrow) and hyperkeratosis
(hk). (d) RCM image (0.5 × 0.5 mm) at suprabasal epider-
mal layer displays keratinocyte disarray (kd). (e) RCM
image (0.5 × 0.5 mm) at the upper dermis shows the pres-
ence of polymorphous vessels (red arrowheads)
P. F.-C. Serrano et al.

383
accuracy in melanocytic skin tumors with non-­
invasively methods has led to the development
and investigation of new imaging tools such as
high-resolution ultrasound, optical coherence
tomography, spectroscopy and RCM. RCM is the
most promising for non-invasively examining
skin structures at a level that allows cellular details
and, such as dermoscopy, it may be useful for dif-
ferential diagnosis between pigmented lesions.
Seborrheic Keratosis (Fig. 22.4)
Seborrheic keratosis is a common benign epider-
mal proliferation. Sometimes this tumor can be
pigmented and then it may be important in the dif-
ferential diagnosis with melanocytic lesions such
as melanoma. On RCM, we can see epidermal pro-
liferation without keratinocytic disarray and long,
parallel, geographic dermal papillae (Fig. 22.4b, d),
which are well delimited by high bright rings of
basal keratinocytes (edge papillae). This architec-
ture pattern has interpretation as benign pattern.
Additionally comedo-like openings filled with
keratin (Fig. 22.4c) and millium cists (Fig. 22.4b)
are visualized. In the case of clonal seborrheic ker-
atosis, a clod pattern resembling melanocytic
lesions may also be visualized [42].
Dermatofibroma (Fig. 22.5)
Dermatofibroma (DF) is a common benign der-
mal proliferation of histiocytes that can arise at
a
c
d
b
500 mm
250 mm
250 mm
Fig. 22.4  Pigmented
seborrheic
keratoses.
(a)
Dermoscopy reveals a star-shaped lesion with sharply cir-
cumscribed and moth-eaten edges, also containing a cen-
tral hyperpigmented area with millium-like cists. (b)
RCM Mosaic (3 × 3 mm) at dermo-epidermal junction
displays geographic dermal papillae and opening full of
keratin material (red asterisks) corresponding to comedo-­
like openings and well-defined, intensely bright, rounded
areas (red arrows) corresponding to millium cysts. (c)
RCM image (0.5 × 0.5 mm) within the mosaic b displays
irregular dermal papillae in size and shape with curved,
invaginated borders (asterisks). (d) RCM image
(0.5 × 0.5 mm) (red dashed line)
22  Reflectance Confocal Microscopy in Oncological Dermatology

384
any age and equally in male and female, but is
more typically seen in young adult women.
Darkly pigmented lesions can simulate dysplastic
nevi or melanoma. Reflectance confocal micro-
scopic examination of DF shows the presence of
a normal epidermis with homogeneously bright
papillary rings and refractile keratinocytes above
the dermo-epidermal junction (Fig. 22.5b) corre-
sponding histologically to the pigmentation of
the basal layer. Moreover, the dermis presents
high refractile, thick collagen bundle correspond-
ing to the sclerotic stroma of the tumor
(Fig. 22.5b). We have not specific images on con-
focal microscopy examination of DF because
spindle fibroblast-like cells, histiocytes and scle-
rotic stroma are too deep, in the reticular dermis.
Although there are not specific images in DF
examination using reflectance confocal micros-
copy, we consider that this technique may be a
useful tool in differential diagnosis with others
cutaneous tumors, particularly when DF is darkly
pigmented and it simulates melanocytic lesions.
Angioma
Angioma is a vascular tumor that consist in
numerous and dilated blood vessels. RCM shows
a
b


Fig. 22.5  Dermatofibroma. (a) Dermoscopy shows a
brown, pigmented network with central scar-like área. (b)
RCM mosaic (5,5 × 5 mm) at dermo-epidermal junction
demonstrates increased density of bright dermal papillary
rings within the complete lesional área. Central portion
shows partly homogeneus and dense collagen bundles
(yellow asterisks). At the periphery, the lesion displays
elongated dermal papillae (red dashed circle)
P. F.-C. Serrano et al.

385
dilated, tortuous blood vessels located at superfi-
cial dermis. As RCM can obtain real time images
we visualize blood flow in these vessels [17].
Pigmented Basal Cell Carcinoma
Pigmented BCC is a clinical and histologic vari-
ant of BCC that may at times be difficult to clini-
cally distinguish from benign pigmented lesions
and melanoma. Agero and co-workers have char-
acterized pigmented BCC with RCM [43]. The
features include the same characteristics previ-
ously mentioned for BCC. Melanin pigment was
not uniformly distributed throughout the tumor
cords. The variable amounts of melanin present,
together with the melanin distribution within the
tumor nests, determined the variability in bright-
ness
of
tumor
nest
visualized
on
RCM.  Contributing to the pigmentation of the
tumor is melanin pigment in benign epidermal
keratinocytes, in tumor cells of BCC, in melano-
cytes interspersed among the tumor cells, and in
melanophages in papillary dermis. Melanin and
melanosomes provide strong contrast with the
surrounding edematous or mucinous dermal
stroma of BCCs under RCM.
Pigmented Mammary Paget Disease
RCM has also been used to study Paget disease
(PD), probably because it has proven to be an
excellent tool for identifying pagetoid spread in
lentigo maligna melanoma and superficial spread-
ing melanoma [44]. In 2007, Longo and co-work-
ers published a case of pigmented mammary PD
with RCM findings suggestive of melanoma [45]:
pagetoid infiltration by large, round atypical cells
with a bright cytoplasm and a dark nucleus
together with numerous large cells with long den-
dritic branches in the stratum corneum and ill-
defined papillary rings. More recently Guitera and
colleagues [46] published a series of nine cases of
extramammary PD and one case of mammary
PD. All the lesions displayed large Paget cells dis-
tributed either individually or in small nests,
which is a characteristic finding in PD. The Paget
cells in the stratum granulosum were visualized as
dark or hyporeflective structures. In four cases,
the cells were seen as bright structures with a dark
halo (targetoid lesion). This halo may correspond
to the cleft-like separation of Paget cells from the
epidermis that is seen on histology and is possibly
due to mucin secreted by these cells or to intercel-
lular fluid. While mucin appears hyporeflective in
RCM images, Paget cells are frequently seen as
hyperreflective, probably due to the reflection of
light from the secretory granules and to the prom-
inent Golgi apparatus and numerous free ribo-
somes that these cells contain. An atypical or
disarrayed honeycomb pattern indicating disrup-
tion of the epidermis is seen in many cases. The
blood vessels are generally abundant, small, and
vertically oriented, a typical finding for skin in the
anogenital region. Considering the non-specific-
ity of the clinical manifestations of PD and the
fact that this disease can mimic a range of inflam-
matory, allergic, and infectious conditions, RCM
may facilitate earlier diagnosis and faster initia-
tion of appropriate treatment [47]. It can also be
useful for selecting the most appropriate biopsy
site and for mapping out margins prior to treat-
ment [48, 49].
Melanocytic Nevi (Figs. 22.6 and 22.7)
RCM is particularly well suited to the imaging of
melanocytic lesions, since the large amount of
melanin they contain provides very good con-
trast. Accurate characterization of the features of
benign and malignant melanocytic lesions has
important implications for diagnosis and/or
screening of pigmented lesions. RCM features of
common melanocytic nevi have recently been
identified and are characterized by the presence
of a homogeneous population of small monomor-
phous round or oval brightly refractile cells
within which nuclei are centrally positioned
(nevomelanocytes) [50, 51]. In benign nevi both
spinous and granular layers show no alteration,
with normal pattern consisting in homogenous
and well-demarcated keratinocytes. Dermal
papillae are uniformly distributed and circum-
scribed by a ring of refractive monomorphous
cells that correspond to small melanocytes and
basal keratinocytes (melanin-rich cells), without
any cytologic atypia.
Junctional nevi display abundant single cell
proliferation at the DEJ that results in the pres-
ence of single small bright polygonal cells outlin-
22  Reflectance Confocal Microscopy in Oncological Dermatology

386
a
b
c
hf
hf
450 µm
250 µm
Fig. 22.6  Acquired nevus. (a) Dermoscopy reveals a
network pattern. (b) RCM image (0.4 × 0.6 mm) corre-
sponds to the red square of the image c. It reveals single
cell proliferation at the dermo-epidermal junction that
results in the presence of single small bright polygonal
cells outlining the dermal papillae displaying a ringed pat-
tern. A sweat loop (red arrowhead) may be seen. (c)
RCM mosaic (3  ×  4.5  mm) showing a ringed pattern.
Sweat loops (red arrowheads) and hair follicles (hf) are
seen
ing the dermal papillae, generating the so-called
“ringed” pattern (Fig. 22.6) on RCM [52]. Some
junctional nevi also display few nests located at
the tip of the cristae and this aspect shows up as a
“meshwork” pattern on RCM that is made by the
junctional enlargement between the papillae
(Fig. 22.6). In these lesions, the presence of cyto-
logic atypia is uncommon. Intradermal nevi
reveal the presence of the so-called “clod” pat-
tern where several nests fulfill completely the
dermal compartment (Fig. 22.7). Nests into the
dermis can show up as compact dense or as
“dense & sparse” nests [53, 54], where the aggre-
gates are large and made by large roundish cells
outlined by epidermal strands.
Moreover,
a
combination
of
mesh-
work + ringed or clod + meshwork + ringed pat-
terns are often found. Usually the presence of
clod pattern is apparent in the centre of the
lesion corresponding to the clinical palpable
area and to the presence of variable sized der-
mal nests.
RCM is particularly useful in discriminating
these lesions from melanoma since dermoscopy
can be not enough specific thus leading to the
overlooking of these nevi.
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387
Blue nevi appear as a consequence of dermal
proliferation of melanocytes. Since the RCM
allows the visualization only of the upper dermis,
these nevi do not represent the best candidate for
confocal imaging. However, a few case reports
describe the presence of spindled/dendritic mela-
nocytes as a confocal clue that may be useful for
the diagnosis of blue nevi [55].
RCM is useful in assessment small and
medium congenital melanocytic nevi. Dark holes
or fissures, corneal cysts, white papillae (i.e., der-
mal papillae with brighter content than normal
skin, without apparent discrete nests or cell) and
dense clusters expanding dermal papillae are
confocal features suggestive of congenital mela-
nocytic nevi [56].
Dysplastic Nevi
RCM features of dysplastic nevi are in contrast to
others melanocytic nevi. As previously men-
tioned, common and junctional melanocytic nevi
preserve the epidermal architecture and they are
a
c
d
b
450 µm
250 µm
hf
*
*
sf
sf
Fig. 22.7  Dermal nevus. (a) Dermoscopy reveals a clod
pattern. (b) RCM image (0.55 × 0.5 mm) at the upper epi-
dermis showing a typical honeycombed pattern. Poligonal
keratinocytes are seen between skin folds (sf). (c) RCM
image (0.55 × 0.5 mm) from exactly the same skin area at
the dermoepidermal junction level reveals the presence
dense nests (yellow asterisks). (c) RCM image (3 × 4 mm)
discloses dense (yellow asterisks) and sparse (white
asterisks) dermal nests, hair follicles (hf) and other annex-
ial structures (red arrows)
22  Reflectance Confocal Microscopy in Oncological Dermatology

388
characterised by a symmetrical architectural pat-
tern and homogeneous cytological characteristics.
On the contrary, dysplastic nevi share features
with common nevi and melanoma [50]. Dysplastic
nevi also show a greater variety in nevomelano-
cyte refractivity, size and shape (nevomelanocytic
population
more
heterogeneous).
However,
nevomelanocytic cells tend to be round or oval as
in common nevi, rather than dendritic as in mela-
nomas [50, 51]. Atypia is seen at dermoepidermal
junction as big isolated, epithelioid cells with
peripheral nucleus [52]. Cell nests may be less
demarcated. Keratinocytes focally lose their
demarcations at the spinous layer associated with
dendrites and occasionally “melanin dust” (high
refractive granular particles that probably repre-
sent melanin bodies). Especially on the face, atyp-
ical nevi may show focal disruption at
dermo-epidermal junction (focal loss of the cell-
cell keratinocytic borders), with low homoge-
neous dermal papillae with absence of demarcation
by refractive cells. Lovatto et al. showed the use-
fulness of RCM in conjunction with digital fol-
low-up in atypical mole syndrome patients,
increasing the accuracy in the assessment of
equivocal lesions and decreasing the number of
benign lesions excised [57].
Spitz/Reed nevi are a family of lesions that
vary in terms of clinical-dermoscopic aspect, his-
tologic features and biologic attitudes. Confocal
microscopy allows the analysis of the superficial
dermis and thus, the exploration of the deeper part
is not possible, hampering the evaluation of some
relevant aspects such as maturation [58, 59].
Although the confocal features of Spitz nevi
are partially characteristic, they remain in most
cases, indistinguishable from melanoma. A good
correlation was found for some histopathologic
aspects and RCM features; some of which are
considered characteristic of Spitz nevi, such as
sharp lateral demarcation and presence of spin-
dled cells. Other correlates that are not very spe-
cific include pagetoid infiltration, junctional and
dermal nests, parakeratosis, trans-epidermal mel-
anin elimination, and inflammatory infiltrate rich
in melanophages. The frequent presence of fea-
tures suggestive of malignancy in Spitz nevi,
such as pagetoid and atypical cells, non-edged
papillae, dishomogeneous nests, and the impos-
sibility to explore the deeper parts of a given
lesion, hamper a reliable diagnosis with
RCM. According to a study conducted by Guida
and co-workers [60], RCM may be useful in dif-
ferentiating melanoma from Spitz nevi with star-
burst or globular pattern on dermoscopy. In this
case, the presence of striking cell pleomorphism
within the epidermis, widespread atypical cells at
the dermoepidermal junction and marked pleo-
morphism within nests were significantly associ-
ated with the diagnosis of melanoma, while
spindled cells and peripheral clefting were found
exclusively in Spitz naevi.
Malignant Melanoma (Fig. 22.8)
Gerger and co-workers demonstrated the appli-
cation of RCM to diagnostic classification of
benign and melanocytic skin tumors [61]. They
consider the images derived from dermo-epider-
mal junction more useful for diagnosis. The
most useful parameters were melanocytic cyto-
morphology and architecture and keratinocyte
cells edge assessment, with high inter-observer
and intra-­observer agreement. On the other hand,
evaluation of melanocytic cell brightness and
dendrite-like structures had less to none diagnos-
tic importance. In this study, observers without
specific dermatopathology experience performed
better than trained dermatopathologists, indicat-
ing that micromorphologic features relevant in
paraffin histology cannot directly be related to
RCM images. In their opinion, an unprejudiced
approach of the observer seems to be more suc-
cessful with respect to diagnostic accuracy. More
recently, a diagnostic semi-quantitative algo-
rithm for RCM evaluation of clinically and der-
moscopically equivocal melanocytic lesions has
been proposed by Pellacani and co-workers
showing a total score ranging from 0 to 8 [62].
This algorithm has two major and four minor
RCM criteria associated to malignancy. Major
criteria (scored two points in the diagnostic algo-
rithm) are the presence of non-edged papillae
and cytological atypia (Fig. 22.8b, c), whereas
the minor criteria (scored one point) are the pres-
P. F.-C. Serrano et al.

389
a
b
c
d
500 µm
300 µm
100 µm
Fig. 22.8  Melanoma. (a) Dermoscopy reveals a struc-
turless, broken network with thickened areas and a few
rosettes. (b) RCM mosaic (6 × 4 mm) at dermoepidermal
junction displays a multicomponent architecture with
ringed (blue dashed circle) and meshwork (yellow
dashed circle) patterns. Red rectangle corresponds to the
magnified image c. (c) RCM submosaic (1.2 × 1.2 mm) of
obtained from mosaic b denotes the presence of edge and
non-edge (red dashed circle) dermal papillae with
numerous atypical cells (red arrows). (d) RCM image
(0.5 × 0.5 mm) at the suprabasal level reveals large round-
ish pagetoid cells (red arrowheads)
22  Reflectance Confocal Microscopy in Oncological Dermatology

390
ence of round, bright, and nucleated cells within
the epidermis (Fig. 22.8d), pagetoid cells wide-
spread throughout the lesion, and cerebriform
clusters and bright nucleated cells within the
dermal papilla. Lesions with a score equal to or
greater than three were quantified for final scores
of 97.3% sensitivity and 72.3% specificity.
Infiltration of the epidermis by melanocytes
(pagetoid melanocytosis) is considered a rele-
vant criterion for melanoma by pathologists,
although sporadically observable in benign
melanocytic lesions. 35 RCM enables the visual-
ization of superficial layers at cellular-level reso-
lution; pagetoid melanocytosis may be exposed
by this technique indicating that melanoma diag-
nosis should be considered, whereas it cannot be
excluded in the absence of pagetoid cells, which
are absent in at least 10% of malignant lesions
[63]. Similar results could be obtained in eczem-
atous lesions, where bright dendritic cells
located
at
the
epidermis
correspond
to
Langerhans cells [64]. Thus, although the pres-
ence of pagetoid dissemination is relevant,
observation of round pagetoid cells is more spe-
cific and it can be considered a further criterion
for melanoma (Fig. 8c). Recently, the validity of
the algorithm was blindly tested on a larger pop-
ulation of equivocal melanocytic lesions, show-
ing in a reproducible clinical setting a 92%
sensitivity and 70% specificity [44]. Segura et al.
developed a two-step method for the diagnosis
of melanoma by RCM [65]. In a preliminary
study, they evaluated 154 skin tumors, including
100 melanocytic and 54 non-melanocytic
lesions, by RCM before their excision. They
observed four confocal features that differenti-
ated melanocytic from non-melanocytic lesions:
cobblestone pattern of epidermal layers, paget-
oid spread, mesh appearance of the dermoepi-
dermal junction, and the presence of dermal
nests. Within melanocytic lesions, the presence
of roundish suprabasal cells and atypical nucle-
ated cells in the dermis was associated with mel-
anoma, and the presence of edged papillae and
typical basal cells was associated with nevi.
Furthermore, a recent study [66] tried to deter-
mine whether specific histological features in
dysplastic nevi had reliable correlates on confo-
cal microscopy and developed an in vivo micro-
scopic grading system, which consists in a
simplified algorithm to distinguish dysplastic
nevi from melanoma and non-dysplastic nevi.
Sixty melanocytic lesions with equivocal derma-
toscopic aspects were analyzed by RCM and his-
topathology, and they found good correlation
between their features. As observed by RCM,
dysplastic nevi were characterized by a ringed
pattern, in association with a meshwork pattern
in a large proportion of cases, along with atypi-
cal junctional cells in the center of the lesion and
irregular junctional nests with short interconnec-
tions. The simultaneous presence of cytological
atypia and of atypical junctional nests (irregular,
with short interconnections, and/or with nonho-
mogeneous cellularity) was consistent with his-
tological
dysplasia,
whereas
widespread
pagetoid infiltration, widespread cytological
atypia at the junction, and non-edged papillae
suggested melanoma diagnosis. Interestingly,
several confocal features for melanoma may be
also identified in amelanotic melanomas. It is
possible to detect melanocytes in a clinically
amelanotic tumor due to the presence of some
melanin inside the pre-­melanosomes, which
have similar size to the wavelength that we use
for imaging. Considering that amelanotic tumors
do not present a high refractivity as other heavily
pigmented lesions, they may be more easily
diagnosed with RCM than with dermoscopy,
which suggests a better early diagnosis of cer-
tain clinically difficult pigmented lesions such as
amelanotic melanoma [67]. In fact, Losi et  al.
described the presence of hyporreflective paget-
oid cells as a clue present in the majority of
amelanotic melanomas (85%) [68].
Melanoma Subtypes
In superficial spreading melanomas (SSM) paget-
oid spread is a hallmark and it is characterized by
the presence of variably shaped bright melanocytes
with focal or widespread distribution. Pagetoid
spread is also commonly observed in lentigo-
maligna type [45, 50], especially around the hair
follicles. Conversely, pagetoid cells are quite rare
in “pure” nodular melanoma whereas ulceration is
a common finding at the epidermal level [31].
P. F.-C. Serrano et al.

391
RCM highlights different shapes of melano-
cytes scattered throughout the epidermis at vari-
ous levels in melanoma. Pagetoid cells usually
have a round shape with bright granular cyto-
plasm and hyporefractive nucleus but can display
a dendritic shape with a barely visible body and
variably long branching terminal structures. A
clear-cut distinction between dendritic-shaped
melanocytes and Langerhans cells has not yet
been established [51].
In MMs, especially in superficial spreading
and lentigo maligna, the DEJ is characterized by
ringed and/or meshwork architecture with large
and bright pleomorphic (“atypical”) cells in early
MMs. The cells can show the tendency to form
nests that can still be compact or can be loosely
arranged, depending on tumor progression. In
early lesions it is a common finding to observe
alteration or abrupt interruption of the normal
DEJ architecture due to flattening of rete-ridges
resulting in irregularly shaped and not well-­
defined papillae (non-edged papillae) [11] or dis-
appearance of dermal papillae substituted by
strands of atypical cells. In advanced stage of
SSM, the massive tumor proliferation occupies
the entire junction and can push up into the epi-
dermal layers as loose aggregates of bright, large
and pleomorphic melanocytes. In nodular mela-
nomas, RCM shows the presence of so-called
“cerebriform” nests [12, 13] that consist of small
and hyporefractive cells forming a large tumor
mass separated by dark thin fissures and bright
collagen septae.
Lesions with regression represent an impor-
tant indication for RCM.  When we deal with
lesions that are exclusively characterized by blu-
ish to greyish granules, the differential diagnosis
includes lichenoid-keratosis or a regressive mela-
noma. RCM is a powerful tool since it permits to
discriminate between the former, showing only a
florid inflammatory infiltrate, and the latter con-
stituted by atypical melanocytes.
Besides its potential diagnostic capability,
RCM has shown promise as a guide for biopsy
collection in the most relevant lesion area [69]; to
monitor the response to treatment [70]; and to
delineate the extension of a lesion before pro-
ceeding to surgical excision [70, 71]. It may also
serve to study possible prognostic features
depending on the correlation of specific RCM
features with superficial spreading or nodular
areas in malignant melanoma. One of the main
drawbacks of RCM is a certain inability to reach
deep tissue in the dermis, reaching a maximum
depth of 250 μm [14]. This limiting factor pre-
vents imaging of structures located deep in the
dermis, especially in cases of hyperpigmented
lesions. In these cases, there is strong attenuation
of the contrast due to massive absorption and
light scattering going through those structures.
The presence of inflammatory cells and collagen
bundles may also diminish contrast and difficult
melanocyte visualization.
Other Reflectance Confocal
Microscopic Applications
in Dermatological Oncology
Dermoscopy-RCM Correlation
Precise dermoscopy-RCM correlation of mela-
nocytic lesions has several potential applications.
First, it enables the study of the specific dermo-
scopic structures [50]. RCM may have an added
value in refining the classification of dermoscopic
structures [72]. On the other hand, RCM may be
useful in differentiation between benign lesions
and those suggestive of malignancy in areas of
equivocal dermoscopic findings [50, 72]. Due to
close relationship between early diagnosis and
life prognosis of the patient, RCM may be par-
ticularly useful in order to increase clinical and
dermoscopic sensitivity. Pellacani et  al. [73]
studied the impact of RCM in the routine mela-
noma workflow. They reported that systematic
application of RCM for equivocal lesions saved
over 50% of benign lesions from unnecessary
excision. The number of lesions needed to excise
a melanoma was 6.8 with RCM examination,
compared with a hypothetical 14.6 without RCM
evaluation.
RCM can be used to evaluate tissue architec-
ture and cytomorphological features, such as pag-
etoid spread that may evade dermoscopic
detection. The level of dermo-epidermal junction
22  Reflectance Confocal Microscopy in Oncological Dermatology

392
is the most useful for the correlation of dermo-
scopic global pattern with the RCM mosaic image
[50]. The dermoscopic appearance of a network
has been correlated to pigmented keratinocytes of
the basal layer surrounding dermal papillae.
Atypical pigment network can result from an
architectural disarray of the basal layer around
papillary dermis. Atypical melanocytes could be
seen in melanomas. Dermoscopic globules were
correlated with nest of melanocytes at the dermo-
epidermal junction or the papillary dermis. Each
globule on dermoscopy could be identified as a
cluster of bright cells on RCM. Peripheral streaks
were correlated with aggregates of elongated
cells, well delineated from the bordering dermis
and contiguous with the central mesh of the
lesion. Some aggregates appeared to be round,
curving around existing dermal papillae and pro-
ducing a pseudopod-like appearance. Other
aggregates produced an arrow-­like arrangement,
representing more linear streaks [50, 72].
RCM is limited in identifying compact ortho-
keratosis and the pigmented structures in the
deeper dermis reported in the histopathological
correlation of the dermoscopic blue whitish veil.
However, RCM permits a complementary hori-
zontal overview of the architectural arrangement
at the dermo-epidermal junction level and clearly
showed focal disruption of the arrangement of
the basal cells, with bright, plump cells compati-
bles with melanophages and of some melano-
cytes in the superficial dermis [50].
Guide to Perform a Biopsy
RCM can help to guide clinicians to perform a
biopsy on a focused area that shows concerning
confocal findings [22, 23, 25, 74].
Margin Assessment and Adjunct
to Surgery
RCM has the potential to define lesion margins
before surgical o nonsurgical therapy. This could
be particularly helpful in margin assessment of
tumors with radial growth phases, including len-
tigo maligna melanomas [70] or some basal cell
carcinomas [75], or tumors that are difficult to
see clinically, such as amelanotic melanomas
[71] or sclerosing infiltrative basal cell carcino-
mas [22]. The main limiting factor to date has
been the limited depth of penetration, which pre-
vents accurate imaging at depths below superfi-
cial dermis. Lack of contrast can also pose a
problem, which might be solved by the future
development of exogenous contrast agents [76].
Despite present limitations, RCM can help iden-
tify areas that are atypical and need biopsy to pro-
vide histologic confirmation.
RCM may aid in the rapid establishment of
tumor margins by examining excised specimens
during procedures such as Mohs micrographic
surgery [75, 77]. Mohs micrographic surgery is a
histologically guided method of removing skin
cancers that maximally preserves normal tissue.
Thin layers of tissues are removed, and the mar-
gins are examined histologically to determine if
cancerous cells remain. Selective removal of
residual tumor is performed until tumor-free mar-
gins are obtained. This procedure can be time-­
consuming since each excised specimen must be
processed by frozen histopathology requiring
embedding, sectioning, and staining. With RCM
of ex vivo, unprocessed tissue, it is possible to
detect neoplastic cells by using 5% acetic acid
and cross-polarized illumination. This technique
makes the neoplastic nuclei more bright and sur-
rounding dermis dark. Recently, it has been dem-
onstrated that RCM may be useful to examine
nonmelanoma skin cancers in ex vivo tissue dur-
ing Mohs micrographic surgery without frozen
sections [75, 77]. This new use of RCM offers the
advantage of rapid visualization of margins com-
pared to permanent sectioning and the ability to
do special stains or permanent stains in areas that
are equivocal. RCM has also been used in vivo
during Mohs surgery to help to locate tumor and
rapidly visualize the margins for BCC and mela-
noma. Flores and co-workers used in vivo RCM
during BCC Mohs surgery prior application of
aluminum chloride for nuclear contrast with
promising results [78]. In addition, Guitera et al.
[79] research found RCM useful in delimitating
lentigo maligna extension.
P. F.-C. Serrano et al.

393
Use of RCM for Evaluating Response
to Treatment
The gold standard for the treatment of several
types of skin cancer is surgical removal (exci-
sional biopsy) of the affected area and the adjoin-
ing healthy tissue to prevent the escape of tumor
cells. However, this is often traumatic, particu-
larly if the affected area is a cosmetic hotspot,
e.g., the face. Excisional biopsy is often preceded
by one or several incisional biopsies aimed at
characterizing the tumor, determining malig-
nancy, and helping the medical practitioner make
an informed decision on the most appropriate
treatment. Furthermore, excisional surgery needs
to be followed up as well and may require addi-
tional incisional surgeries. With these problems
in mind, the development of non-invasive treat-
ment protocols has been important. Major break-
throughs are the uses of photodynamic therapy
(PDT) and imiquimod application.
Photodynamic Therapy
RCM Monitoring of the Response of BCC
to PDT Treatment
MAL-PDT treatment has been very successful
in clinical trials (reviewed in [80]). Different
histological subtypes of BCC have been treated
with PDT.  Several studies have addressed the
potential of RCM to follow up the response of
BCC lesions to PDT using MAL [81]. Venturini
et  al. [82] reported the usefulness of RCM in
detecting early recurrences of BCC after MAL-
PDT and even by means of RCM, dermoscopi-
cal false positive of BCC recurrence after
MAL-PDT treatment may be ruled out. Gorlin
syndrome (GS) and xeroderma pigmentosum
(XP) are congenital skin diseases that share a
genetic predisposition to develop skin cancer.
Patients suffering from these genodermatoses
often develop BCC.  One recent study used
MAL-based (Metvix®) PDT in a small group of
GS and XP patients and followed up tumor cell
clearance by RCM. Under these conditions, this
study reported excellent results in the use of
RCM to determine the degree of clearance after
PDT [81].
Treatment of Actinic Keratosis with PDT:
RCM Follow-Up
5-ALA PDT is rapidly becoming a reference
treatment in the management of actinic kerato-
sis (AK) [83]. In addition to close to 100%
efficacy, the cosmetic outcome of the proce-
dure is vastly superior to that of surgery. An
area of particular interest is the management
of skin cancer, mainly SCC, of which AK is
considered a direct precursor [84] in immuno-
suppressed individuals, e.g., those undergoing
solid organ transplantation. In this context, a
recent study has evaluated, using RCM, the
response to 5-ALA PDT in organ transplant
recipients (OTR) that display AK.  In this
study, RCM proved to be an excellent tool to
monitor the response to PDT [85]. A recently
published article reported that RCM was use-
ful in monitoring the efficacy of daylight-PDT
in AK [86].
Imiquimod Treatment
RCM Monitoring of the Response of BCC
to Imiquimod
In 2003, our group reported the use of RCM for
the diagnosis of BCC and for monitoring BCC
clearance following the application of imiquimod
[87]. In 2004, another study used RCM to deter-
mine the effect of imiquimod treatment on BCC
prior to Mohs microsurgery [88]. This study sug-
gests that RCM can be used to monitor the
response of a lesion to imiquimod, aiding in the
patient’s and dermatologist’s decision on whether
surgery is further needed.
RCM Monitoring of the Response
of Actinic Keratosis to Imiquimod
The applicability of RCM to evaluate the response
of AK to imiquimod has been assessed in a small
group of patients with subclinical features of AK
[89]. Another study compared vis-a-vis the effect
of imiquimod (three times/week for 4 weeks) and
5-ALA PDT in AK in organ transplantation
patients [85]. This study was used to generate a
table of RCM-visualized parameters and their
usefulness for diagnosis of AK and for monitor-
ing its response to imiquimod or PDT.
22  Reflectance Confocal Microscopy in Oncological Dermatology