Review

Dermatology 2005;211:5462 DOI: 10.1159/000085581

Trichoscan: What Is New?

Rolf Hoffmann
Dermaticum, Practice for Dermatology, Freiburg, Germany

Key Words Hair growth measurement Alopecia Computer analysis Mesh terms Epiluminescence microscopy Androgenetic alopecia

Abstract The treatment of androgenetic alopecia (AGA) is usually long lasting, and the effects of treatment attempts are difcult to measure. Consequently, there was a need for a sensitive tool to monitor hair loss and treatment response. Therefore, we developed the Trichoscan as a method which combines epiluminescence microscopy with automatic digital image analysis for the measurement of human hair. The Trichoscan is able to analyze all important parameters of hair growth (density, diameter, growth rate, vellus and terminal hair density) with an intraclass correlation of approximately 91% within the same Trichoscan operator and an intraclass correlation of approximately 97% for different Trichoscan operators. The application of the technique was demonstrated by comparison of the hair parameters in 9 men with frontal balding which were treated for 6 months with 5% minoxidil. Even in this small cohort of patients, we noticed after 3 months of treatment compared to baseline a signicant increase in hair density (+21.3 hairs/cm2; p = 0.047) and cumulative hair thickness (+0.61 mm; p = 0.008) and after 6 months a signicant increase in hair density (+34 hairs/cm2; p = 0.011) and cumulative hair thickness (+0.88 mm; p = 0.010). The study

shows that the Trichoscan has many advantages. It can be used for clinical studies to compare placebo versus treatment or to compare the relative potencies of different hairgrowth-promoting substances. It can be used for studying AGA or other forms of diffuse hair loss, and it can be adopted to study the effect of drugs or laser treatment on hypertrichosis or hirsutism. The drawbacks, however, are that the Trichoscan still needs a hair dye for contrast enhancement and the measurement area must be clipped before analysis. This mini-review summarizes recent attempts to optimize the technique and shows new options such as the calculation of follicular units or the anagen hair count.
Copyright 2005 S. Karger AG, Basel

Introduction

Hair loss or hair thinning is a common complaint in clinical dermatology. In established cases of androgenetic alopecia (AGA), the characteristic hair loss patterns are easily discernible. However, especially with females, the clinician is often challenged by patients with initial stages of AGA where hair loss is reported, but alopecia is not recognizable, or the effects of treatment attempts are difcult to measure. Quantitative methods for the analysis of human hair growth and hair loss are necessary to determine changes in hair growth with disease progression and the efcacy of hair-growth-promoting drugs. While re-

2005 S. Karger AG, Basel 10188665/05/21110054$22.00/0 Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Accessible online at: www.karger.com/drm

Rolf Hoffmann, MD Dermaticum, Practice for Dermatology Kaiser-Joseph-Strasse 262 DE79098 Freiburg (Germany) Tel. +49 761 383 7400, Fax +49 761 383 7401, E-Mail rolf.hoffmann@dermaticum.de

viewing the capabilities of the different analysis methods, the common theme emerges that most techniques are of little use to the clinician because they are time consuming and often costly or difcult to perform [1, 2]. Therefore, an operator- and patient-friendly, inexpensive, validated and reliable hair growth evaluation method is a rational need. Such a method should be able to analyze the important parameters of hair growth, which are: (1) hair density (n/cm2), (2) hair diameter (m), (3) hair growth rate (mm/ day) and (4) vellus/terminal hair density.

Trichoscan

Recently we have described Trichoscan [3, 4] as a method which combines standard epiluminescence microscopy (ELM) with automatic digital image analysis for the measurement of all important hair parameters in situ. For a Trichoscan analysis to be conducted, a transitional area of hair loss between normal hair and the balding area must be chosen and clipped. During clinical trials, all clipped areas must be landmarked with a central, single red tattoo which serves as a visible reference point throughout the study. Gray or fair hairs have only limited contrast in comparison to the scalp. Therefore, the clipped hairs within the target area are dyed with a commercially available solution (Goldwell 2N schwarz, Darmstadt, Germany). The approach to dye the hairs for hair growth studies has been described to give the same results as that with uncolored hairs [5]. Thereafter, the colored area is cleaned with an alcoholic solution (Kodan Spray, Schlke & Mayr, Vienna, Austria) and, while the area is still wet, digital images are obtained either with digital video dermoscopes at 20-fold (analyzed area: 0.62 cm2) and 40fold (analyzed area: 0.23 cm2) magnication by means of a digital ELM system (Fotonder Derma, Teachscreen Software, Bad Birnbach, Germany) or with digital cameras such as Nikon Coolpix 4500, Coolpix 8400 or Canon Powershot A95 (analyzed area: approx. 1.5 cm2; g. 1). All systems are equipped with a rigid contact lens which ensures that the images are always taken at the same distance from the scalp. Due to the fact that the camera must be pressed onto the scalp, the hairs are always attened relative to the skin and camera. Images are usually taken on day zero immediately after clipping and after different time points according to choice. For the measurement of hair density (n/cm2), hair diameter (m), hair growth rate (mm/day) and vellus/terminal hair density, the Trichoscan software has been developed to analyze these parameters (g. 2). The software

is provided with the scalp hair images, and for each image it works step by step through the following algorithms: (1) selection of the color component; (2) artifact rejection (bubbles and reections); (3) determination of threshold; (4) thresholding; (5) labeling/identication of hair regions (hair bers); (6) deselecting of small regions (smaller than minimal hair length); (7) analysis of each hair ber region: (a) search for the longest straight line (fullling several predened conditions) at the edge of the analyzed hair region and (b) reduction of hair region of detected hair; (8) repetition of steps 8a and 8b until no more hair is found; (9) repetition of analysis of all hair regions (10) calculation of the number of hairs, hair density and mean/median hair thickness/sum of hair thickness. The software was validated by use of more than 500 images, which were taken from study participants. The algorithm excludes air bubbles, dust, small hemangiomas, nevi, scales etc. from the calculation without interfering with the number of detectable hairs. The detection limit of the software depends on the resolution (pixels) of the digital cameras. Using a video system, hairs smaller than 14 m cannot be analyzed, whereas with higherresolution 7-megapixel cameras hairs 6 m thick can be detected.

Trichoscan: What Is New?

New Data with 5% Minoxidil (Regaine) The application of the Trichoscan technique is demonstrated with new data from 9 men with frontal balding (AGA) evaluated after 3 and 6 months of treatment with 5% minoxidil. All patients had had recognized progressive thinning of hairs and hair loss for more than 2 years. All presented the clinical nding of mild to moderate AGA with various degrees of involvement, but all had frontal balding. Subjects with other forms of alopecia were excluded from the study. All patients were treated in a nonblind fashion with Regaine (5% minoxidil) for 6 months. All actively treated patients had had no treatment whatsoever for hair loss for at least 1 month before initiating this trial. Images were taken on day zero immediately after clipping the hair, and then 3 and 6 months after the initial visit. For all hair parameters the differences between the results after 3 and 6 months and compared baseline were calculated. A Students t test was used to calculate whether the results were statistically signicant. Using this approach we noticed after 3 months of treatment a signicant increase in hair density (+21.3 hairs/cm2; p = 0.047)

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Fig. 1. Example of the Trichoscan equipment with Canon Powershot A95. This tool allows an area of 1.5 cm2 to be analyzed.

Fig. 2. Example of the Trichoscan analysis for total vellus and terminal hair number, total vellus and terminal

hair density. The gure illustrates a digital image taken at 20-fold magnication and shows the area of 0.65 cm2 (blue circle) which is analyzed with the Trichoscan software. The Trichoscan results are illustrated on the right side, where the detected hairs are illustrated with different colors. Yellow hairs touch the borders of the circle. The right lower part of the gure shows a histogram of the different hair lengths detected by the Trichoscan software. The red tattoo is not measured, due to the fact that the software is color calibrated and ignores red colors.

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Fig. 3. Hair counts and cumulative hair thickness were analyzed for 3 and 6 months in 9 men with frontal balding (vertical lines indicate 95% condence intervals). For all hair parameters, the differences between the results after 3 and 6 months compared to baseline were calculated. A Students t test was used to calculate whether the results were statistically signicant. Using this approach we noticed after 3 months of treatment a signicant increase in hair density (+21.3 hairs/cm2; p = 0.047) and cumulative hair thickness (+0.61 mm; p = 0.0084). After 6 months, we also noticed a signicant increase in hair density (+34 hairs/ cm2; p = 0.011) and cumulative hair thickness (+0.88 mm; p = 0.0098).

250

200

150

100 0 3
Time (months)

2 6

200 Terminal hair density (1/cm)

20 Vellus hair density (1/cm)

180

15

160

10

140

120 0 3 Time (months) 6

0 0 3 Time (months) 6

Fig. 4. Terminal hair counts were analyzed for 3 and 6 months in 9 men with frontal balding. Interestingly, the increase in overall hair density was mainly due to hairs thicker than 40 m (terminal hairs, +23.28 hairs/cm2 after 3 and +32.46 hairs/cm2 after 6 months).

Fig. 5. Vellus hair counts remained unchanged during the 6-month

treatment of 9 men with frontal balding (vellus hairs, 1.96 hairs/ cm2 after 3 and +1.52 hairs/cm2 after 6 months).

and cumulative hair thickness (+0.61 mm; p = 0.0084). After 6 months, we noticed a signicant increase in hair density (+34 hairs/cm2; p = 0.011) and cumulative hair thickness (+0.88 mm; p = 0.010; g. 3). Interestingly, the increase in hair density was mainly due to hairs thicker than 40 m (terminal hairs, +23.28 hairs/cm2 after 3 and +32.46 hairs/cm2 after 6 months; g. 4), whereas the vellus hair count remained nearly unchanged (terminal hairs, 1.96 hairs/cm2 after 3 and +1.52 hairs/cm2 after 6 months; g. 5).

Detection of Blond and Gray Hairs without a Hair Dye In preliminary experiments, we tried to analyze fair or gray hair with the Trichoscan software, but these hairs provided only limited contrast. Coloring the hairs resulted in a marked increase in hair detectability and did not interfere with the basic parameters of hair growth. However, this procedure is a bit time consuming, and consequently we are trying to make the Trichoscan software hair dye independent. In some other modications we have tried to digitally increase the contrast. Some manufacturers of optics such

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Cumulative hair thickness (mm)

hair density Cumulative hair thickness

Hair density (n/cm)

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Fig. 6. Using the Dermlite Pro Multispec-

tral, different light colors can be tried for contrast enhancement. This gure shows the results within the same scalp area but with different light sources. These different colors allow us to discriminate hairs better.

Fig. 7. In order to analyze the number of

follicular units per square centimeter, we dene the maximal distance of individual hairs, which belong to one follicular unit (blue circle). With this mathematical approximation we can reveal follicular units, their density and the number of follicular units containing 1, 2, 3 or more hairs. Hairs are marked yellow. A follicular unit is dened with a blue circle. Some units contain only 1 hair, whereas others have more hairs. In the latter case, different blue circles touch each other. Those coalescent blue circles are counted as one follicular unit. This tool may help surgeons to plan a hair transplantation better, in order to avoid overtreatment and to preserve as much donor area as possible.

as the Dermlite Pro Multispectral allow the use of different light colors. Different colors give different contrasts (g. 6) and may be better for hair analysis. Another approach is to rene the software further and to use algorithms which cannot be calculated by older computer chips due to their slower computing capacities. For this purpose, 30 volunteers were recruited, and images were

taken with and without the application of hair dye. These preliminary results show that we are able to quantify even blond hairs without the use of hair dye. This analysis tool currently requires a relatively longer computing time, but with recent developments in computer chip technology, these calculations can be successfully done with all Intel Pentium 4 processors and their equivalent. With rapid

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Day 0, complete clipping

Day 3, after hair dye

Fig. 8. For the anagen hair count, we clip the hairs completely and take an image of the blank area (left image). Three days later, we dye the clipped area, make the image and analyze hair density. As in this example, some hairs do not grow (red arrows) and are too short to be counted. By mathematical approximation these hairs are dened as telogen hairs. Furthermore the camera remembers the time when the images have been taken, and the Trichoscan software calculates automatically the exact time (minutes) between both images. Due to the fact that the Trichoscan also measures hair length, we can easily calculate the hair growth velocity.

advances in computer chip technology, software analysis processing time will be reduced signicantly. Analysis of Follicular Units Human scalp hairs do not grow singly, but rather in groups. These so-called follicular units contain several hairs, and their number is not constant during life. During AGA individual hairs within follicular units miniaturize and eventually cannot be seen macroscopically. As a consequence follicular units may contain 6 hairs in adolescence but only 2 hairs some years later, when AGA has developed. One treatment option for AGA is the replacement of follicular units by transplantation. These transplants are usually taken from the occiput and thus far the experience of the surgeon decided how much donor skin is taken for each hair transplantation. Ideally, however, a surgeon would calculate the number of follicular units necessary in bald regions and in addition calculate the density of follicular units in donor areas. In order to analyze the number of follicular units per square centimeter we dened the maximal distance of individual hairs which belong to one follicular unit. With this mathematical approximation we can reveal follicular units, their density and the number of follicular units containing 1, 2, 3 or more hairs (g. 7). In the future, this tool may help surgeons to plan a hair transplantation better, in order to avoid overtreatment and to preserve as much donor area as possible.

Anagen Hair Count During catagen, hair growth stops, and in telogen there is no longer hair growth but only some hair shaft elongation due to the so-called exogen phase of the hair cycle where the hair is eventually pushed out. In order to measure only those hairs which grow considerably (10.20 mm; anagen hairs) herewith we introduce for the rst time the anagen hair count. For this purpose, we clip the hairs completely and take an image of the blank area (g. 8). Three days later, we dye the clipped area, make the image and analyze hair density. With this approach we encountered several advantages. Firstly, this approach makes the Trichoscan clipping independent as complete clipping is a very easy procedure with common electric hair clippers. Secondly, the camera remembers the time when the images have been taken and the Trichoscan software calculates automatically the exact time (minutes) between both images. Due to the fact that the Trichoscan also measures hair length, we can easily calculate the hair growth velocity. Thirdly, any drug such as nasteride for the treatment of AGA, which increases anagen hairs after time, will result in an increased anagen hair count and this can be measured with the Trichoscan. Fourthly, some therapeutic options for AGA such as minoxidil typically induce an increased shedding of telogen hairs some weeks after application. When total hair counts are calculated, this results in an articial drop of hair density, which recovers when new hairs grow back. With the

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anagen hair count we do not see this effect, as we are able to concentrate on those hairs only which grow considerably (10.20 mm/day, anagen hairs) between clipping and 3 days later. In our view, this approach allows the analysis of the most relevant anagen hairs.

Discussion

Hair diseases such as scarring alopecias, alopecia areata or trichotillomania do not usually need a quantitative method to evaluate the amount of hair shedding. However, AGA, the most common form of hair loss, is difcult to quantify. Although scalp biopsies can be justied in that microscopic examination of scalp skin affected by AGA can identify and quantify any changes resulting from treatment, this invasive technique is often not suitable to monitor patients over a prolonged period of time. As early as 1964, Barman et al. [6] related a method that used optical contact microscopy to calculate these parameters, and much later Hayashi et al. [7] and recently DAmico et al. [8] described a similar approach for the measurement of hair growth by the use of optical microscopy and computer analysis. However, these authors were unable to automate the process of calculation and measured the thickness of hairs visually with a cursor on a computer monitor. The authors calculated that the results from different investigators, but from the same image, differ by 88.4%, which makes such a semiautomatic method unsuitable for clinical practice. A similar approach has been tested with the use of the phototrichogram, which has proven to be a suitable and noninvasive tool to monitor the hair growth phases in situ. This technique has been improved by the image analysis [9] and later with the use of immersion oil and digital contrast enhancement [10]. However, although a marked improvement of the images and more accurate quantitative data were collected, a fully automated analysis is not yet possible to our knowledge as this technique still relies on processing by qualied technicians and computer-assisted image analysis. Nevertheless, the contrast-enhanced phototrichogram [11] is being continuously optimized, and we will see what type of results it will bring. AGA can be dened as an androgen-dependent process in genetically predisposed individuals, where balding is due to miniaturization of affected hair follicles, changing large terminal hair follicles into small vellus-like hairs [1214]. Any successful treatment should therefore stop or reverse the process of hair follicle miniaturization, increase the number of terminal hair follicles whilst reduc-

ing vellus hair counts or increasing the frequency of anagen hairs [15]. This concept is illustrated by the phase III studies of men with AGA treated with nasteride [16]. In these studies, macrophotographs were taken and hairs were counted. This technique produces counts of visible hairs, which means that tiny vellus-like hairs cannot be seen or counted. However, during treatment, these vellus-like hair follicles get thicker and subsequently increase the hair count results when the macrophotograph method is used. A major disadvantage of this technique is that it cannot monitor the expected continuous increase in hair thickness during treatment. As a consequence the phase III studies of men with AGA treated with nasteride revealed that the increase in hair counts reaches a plateau after 1 year of treatment, whereas the hair coverage analyzed by global photographs increased continuously [16]. This increase in hair coverage is due to an increase in hair thickness as shown by histological examination [14], the direct measurement of hair thickness [17] and by the continuous increase in hair weight [18]. Although the Ludwig pattern of AGA in women differs in appearance from the Hamilton pattern occurring in men, these pathophysiological mechanisms seem to be similar, because female AGA patients treated with cyproterone acetate [19] or minoxidil [20, 21] experience an increase in hair thickness or hair weight. Therefore, a reliable hair-counting method should primarily be able to calculate the number and thickness of hairs, which is stable within at least 1 cm above the scalp [22, 23], in a dened area of the scalp. From a clinical perspective, the hair thickness is very important [24], whereas the growth rate (mm/day) and the anagen/telogen ratio are of secondary importance. With the Trichoscan we have created an automated software program for the analysis of the aforementioned parameters of hair growth. Because the described technique is a modied and computerized trichogram we called it Trichoscan. In our view, the Trichoscan has many advantages. Firstly, it is investigator independent. In other studies using the unit area trichogram, a substantial difference between the collected data from different investigators was noted. In these studies a signicantly larger mean total hair count was reported from experienced versus inexperienced observers [25]. Our results show that this is not the case for the Trichoscan technique. Secondly, many methods are not really validated. The hair weight test is a good example where the hair is clipped in a dened target area. However, the sample error for different investigators is unknown. This is mainly due to the methodology, because once the hairs are clipped a second investigator cannot clip the same area again to

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assess the reproducibility of the method. In contrast, the Trichoscan is validated with dened values for intraclass correlation between the same and different investigators. Thirdly, some methods are associated with considerable discomfort to the patient such as the repeated plucking of hairs required by the trichogram technique. The Trichoscan relies on a rather small analyzed area of the scalp, which is barely visible afterwards. The tiny tattoo is the only discomfort patients may recognize. Fourthly, some methods to count hairs are tedious and time consuming. By contrast, Trichoscan can be performed by experienced hands within 812 min hands on. Fifthly, the amount of necessary equipment items is small. Many dermatologists already have ELM systems, and these physicians would only need the Trichoscan software. In summary, with the Trichoscan an automatic software program has been created for the analysis of the most important parameters of hair growth. In small clinical trials with AGA-affected individuals we show that the Trichoscan is able to reveal a response to treatment with 5% minoxidil. The Trichoscan images are taken either with a video or digital system for ELM. These devices produce high-quality and reproducible digital images, because the images are always taken at the same distance of the lens to the skin surface. The drawbacks of this technique are still hair clipping and the use of a hair

dye before the analysis. One of the denitions in the Trichoscan software algorithm is that a hair is straight. This is one of the reasons why scalp hairs must be clipped before the analysis. Clipped short hair stubbles are straight in the software sense. However, in an attempt to analyze body hairs as well, we modied the software, to recognize curved, thin body hairs. First preliminary results show that in less dense areas of hair such as the back, we are able to detect and quantify curved hairs without clipping. Furthermore, the detailed analysis of follicular units will also be possible as a tool for hair surgeons. In the future, digital cameras of 7 or more megapixels will allow a detailed analysis of images with much higher resolution. With different light sources we will try to develop the software such that in the near future a hair dye will no longer be necessary. Furthermore, we are able to analyze larger scalp areas and to calculate the anagen hair count as well as the number of follicular units.

Acknowledgements
The work of U. Ellwanger and H. Ldtke (Datinf GmbH, Tbingen, Germany) in programming the software is gratefully appreciated. All trademarks are the property of their respective owners.

References
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16 Kaufman KD, Olsen EA, Whiting D, Savin R, DeVillez R, Bergfeld W, et al: Finasteride in the treatment of men with androgenetic alopecia. Finasteride Male Pattern Hair Loss Study Group. J Am Acad Dermatol 1998; 39: 578 589. 17 Steiner D, Bedin V, Pasello RR: Hair shaft diameter evaluation in AGA before and after nasteride 1 mg/day. Annu Meet AAD, New Orleans, 2000, poster No 284. 18 Whiting DA: Advances in the treatment of male androgenetic alopecia: A brief review of nasteride studies. Eur J Dermatol 2001; 11: 332334.

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23 Jackson D, Church RE, Ebling FJ: Hair diameter in female baldness. Br J Dermatol 1972; 87:361367. 24 de Lacharriere O, Deloche C, Misciali C, Piraccini BM, Vincenzi C, Bastien P, et al: Hair diameter diversity: A clinical sign reecting the follicle miniaturization. Arch Dermatol 2001; 137:641646. 25 Rushton DH, Unger WP, Cotterill PC, Kingsley P, James KC: Quantitative assessment of 2% topical minoxidil in the treatment of male pattern baldness. Clin Exp Dermatol 1989;14: 4046.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.