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TrichoScan: combining epiluminescence microscopy
with digital image analysis for the measurement
of hair growth in vivo
Hair loss or hair thinning is a common complaint in clinical dermatology,and patients seeking advice for hair loss are not necessarily bald. Also theeffects of treatment attempts are hard to measure. Consequently, there is aneed for a sensitive tool to monitor hair loss and treatment response. Such amethod must be able to analyze the biological parameters of hair growth,which are: 1: hair density (n/cm2), 2: hair diameter (µm), 3: hair growth rate(mm/day) and 4: anagen/telogen ratio. Here we present the TrichoScan as amethod which combines epiluminescence microscopy (ELM) with automa-tic digital image analysis for the measurement of human, and potentiallyanimal hair, in situ. The TrichoScan is able to analyze all biological parame-ters of hair growth with a so-called intraclass correlation of approx. ninety-one percent with the same TrichoScan operator and an intraclass correlationof approx. ninety-seven percent for different TrichoScan operators. Theapplication of the technique is demonstrated by comparison of the hair para-meters in individuals without apparent hair loss with men with untreated AGA and men after treatment with finasteride (1 mg/day), where we were logy, Philipp University, Deutschhaus-straße 9, 35033 Marburg, Germany.
able to detect a significant increase in hair counts and cumulative hair thick-ness 3 and 6 months after treatment. The advantage of the TrichoScan is that it can be used for clinical studies to compare placebo versus treatment or to compare different capacities of different hair growth promoting sub- stances, 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 Net: http://www.med.uni-marburg.de/hautklinik hypertrichosis or hirsutism. (Key words: hair, alopecia, computer analysis.) Hair loss or hair thinning is a common complaint in (mm/day) and 4: anagen/telogen ratio. This paper des- clinical dermatology and patients seeking advice cribes the TrichoScan as such a method which combines for hair loss are not necessarily bald. In established standard epiluminescence microscopy (ELM) with automa- cases of androgenetic alopecia (AGA) characteristic pat- tic digital image analysis for the measurement of human, terns are easily discernible. However, especially in females and potentially animal hair, in situ. The application of the the clinician is often challenged by patients with initial technique is demonstrated by comparison of the aforemen- stages of AGA where hair loss is reported but alopecia is tioned hair parameters of individuals without apparent hair not recognizable or the effect of treatment attempts are loss with men with untreated AGA and men after treat- hard to measure. Consequently, there is a need for a sensi- tive tool to monitor hair loss and treatment response.
Numerous methods have been reported  to assess the rateof hair growth. The techniques can be classified as either Materials and methods
invasive (e.g. biopsies [2, 3]), semi-invasive (trichogram [4,5], unit area trichogram ) or non-invasive (e.g. global Volunteers and patients
hair counts , phototrichogram [8-13]) methods. Quantita-tive methods for the analysis of human hair growth and hair A total of 56 persons (25 females, age range 25-48 years, loss are necessary to determine the efficacy of hair promo- mean 34 years; 31 males, age range 26-39 years, mean ting drugs, and while reviewing the capabilities of the diffe- 32 years) underwent the study. Ten out of fifty-six volun- rent methods, the common theme emerges that most tech- teers (5 females, age range 25-48 years, mean 34 years; niques are of little use to the clinician because they are time 5 males, age range 26-39 years, mean 32 years) were consuming, often costly or difficult to perform [14, 15].
recruited for the initial experiments to analyse the reprodu- Therefore, an operator- and patient-friendly, inexpensive, cibility of the method. The measured parameters were hair validated and reliable method is a rational need.
thickness and hair numbers at the occiput.
Such a method must be able to analyze the biological Seventeen male patients (age range 25-48 years, mean parameters of hair growth, which are: 1: hair density 34 years), who had recognized progressive thinning of hair (n/cm2), 2: hair diameter (µm), 3: hair growth rate and hair loss for more than four years were included to EJD n° 4, vol. 11, July-August 2001 analyse the progression of AGA with and without treat-ment. All patients presented the clinical finding of mild tomoderate AGA with various degrees of involvement, thatwere classified according to the Hamilton scale  (II-V). Subjects with other forms of alopecia were excludedfrom the study. Twelve of 17 male patients with AGAwere treated with Propecia® (1 mg finasteride/day) for sixmonths. All patients actively treated had had no treatmentwhatsoever for hair loss at least one month before initia-ting this study. Eleven of fifty-six healthy male volunteers(age range 28-55 years, mean 36 years) who had experien-ced no episodes of hair thinning or hair loss, recent ill-nesses or general health disturbances, were recruited as acontrol group. Clinical examination revealed no evidenceof any hair disorder with either the female or the malevolunteers. The measured parameters were hair thicknessand hair numbers at the vertex.
For the analysis of daily hair growth and the anagen/telogenratio, 18 additional volunteers with AGA were recruited.
Clipping of hairs
In individuals affected by AGA, a transitional area of hairloss between normal hair and the balding area was definedand an area of 1.8 cm2 was clipped (Hairliner, Wella Ger-many) (Fig. 1A-D). In volunteers without AGA (controls)the vertex was chosen for clipping. All clipped areas weremarked with a central, single black tattoo. The tattoo wasvisible throughout the study. In those 18 volunteers whowere recruited for the analysis of the anagen/telogen ratiothe scalp was clipped at two locations (vertex and occiput)and was analysed by 2 investigators with the TrichoScansoftware. Epiluminescence microscopy (ELM) of clipped hairs
Gray or fair hairs have only limited contrast in comparisonto the scalp. Therefore, the clipped hairs within the targetarea were dyed for 12 min (Fig. 1E-H) with a commer-cially available solution (RefectoCil®, Gschwentner,Vienna, Austria), which is normally used for the coloring Figure 1. This is a stepwise illustration of the complete Tri- of eye brows or lashes. The approach of dyeing the hairs choScan procedure. A: a representative area of the scalp is for hair growth studies has been described as giving the chosen and the plastic template is applied; B: all hairs are same results as uncolored hairs . For the analysis of carefully combed through the plastic template; C: the hairs hair number and thickness the hairs were colored imme- are shaved on the scalp surface; D: the shaved area is diately after shaving and for the analysis of the hair 1.8 cm2 in size; E: 1 cm of dye is applied onto a wooden growth rate and anagen-telogen ratio the hairs were colo- stick; F: 3 drops of developer are mixed (G) with the dye; H: the dye is carefully applied onto the shaved area; I: after Thereafter, the colored area was cleaned (Fig. 1I) with an 12 min the dye is carefully removed with an alcoholic solu- alcoholic solution (Kodan® Spray, Schülke & Mayr, tion; J: digital images are taken at 20- and 40-fold magnifi- Vienna, Austria) and digital images were obtained at 20- cation while the area is still wet. fold (analyzed area: 0.62 cm2) and 40-fold (analyzed area:0.225 cm2) magnification by means of a digital ELM sys- Software for digital image and statistical analysis
tem (Fotofinder DERMA, Teachscreen Software, BadBirnbach, Germany) while the area was still wet (Fig. 1J).
For the measurement of hair density (n/cm2), hair diameter This digital camera is equipped with a rigid contact lens (µm), hair growth rate (mm/day) and anagen/telogen ratio, which ensures that the images are always taken at the software was developed (TrichoScan) to analyze these same distance from the scalp. Due the fact that the camera parameters (Fig. 2). The software works step by step must be pressed onto the scalp, the hairs are always flatte- 1. Selection of color component; 2. Artifact rejection Images were taken at day zero immediately after clipping, (bubbles and reflections); 3. Determination of threshold; 4.
two and three days after clipping, and three and six Thresholding; 5. Labeling – Definition of hair regions; 6.
months after the initial visit, respectively. Two different Deselecting of small regions (smaller than minimal hair investigators each took three images from the same patient length); 7. Tattoo elimination (works by using the fact that the tattoo is a large, dark region located in the center of the EJD n° 4, vol. 11, July-August 2001 Figure 2. Example of theTrichoScan analysis ofhair number, hair density,cumulative hair thicknessand anagen/telogen ratio.
The figure illustrates adigital image taken at 20-fold magnification (leftside of the image) andshows the area of 0.65 cm2(blue circle) which is ana-lyzed with the TrichoScansoftware. The TrichoScanresults are illustrated onthe right side, where thedetected hairs are illustra-ted with different colors.
Red hairs are non-growinghairs (telogen), greenhairs are growing hairs(anagen) and yellow hairstouch the borders of thecircle. The right lowerpart of the figure shows ahistogram of the differenthair lengths detected bythe TrichoScan software. image); 8. Analysis of each hair region: a. Search for the Precision and sensitivity
longest straight line (fulfilling several predefined condi-tions) at the edge of the analysed hair region, b. Reduction The algorithm excludes all air bubbles, dust, small hae- of hair region of detected hair; 9. Repetition of steps 8a and mangiomas, nevi, scales, etc., from the calculation without 8b until no more hair is found; 10. Repetition of analysis of interfering with the number of detectable hairs. In doing all hair regions; 11. Calculation of number of hairs, hair so, only hairs are counted and the precision of the method density, and mean/median hair thickness/sum of hair thick- is therefore approximately 100%. The detection limit of ness. The software was validated by use of more than 500 the software is 5 µm in thickness. Hairs smaller than 5 µm images, which were taken from the study participants.
Measurement of hair thickness and hair number
Total time “hands-on” for TrichoScan operator
In 10 volunteers the hair number (Fig. 3) and cumulativehair thickness (Fig. 4) was analysed in the same area, The complete procedure was finished within 15-20 min.
three times by the same investigator. The percentage of The total time “hands-on” for the TrichoScan operator was variation in hair count between volunteers or so-called approx. eight to twelve min (Fig. 1A-J and Fig. 2).
intraclass correlation, is estimated at 90.9%. The intra-class correlation for the cumulative hair thickness is Effect of the hair dye
In preliminary experiments we tried to analyse fair or gray hair with the TrichoScan software. However, these hairs In 5 volunteers the hair number (Fig. 5) and cumulative produced only little contrast and coloring the hairs resulted hair thickness (Fig. 6) was analysed in the same area once, in a marked increase in hair detectability and did not inter- but by two independent investigators. The intraclass corre- fere with the four basic parameters of hair growth. The lation is estimated for the hair count at 97.6%, for the dye must be applied for 11-13 min. More than 13 min will unintentionally dye the scalp skin. Less than 11 minresults in incomplete staining of hairs.
Analysis of total hair counts and cumulative hair thicknessin volunteers without AGA, with untreated AGA, and AGA Effect of the tattoo
treated with finasterideIn individuals affected by AGA, a transitional area of hair In the experiments presented here we used a single black loss between normal hair and the balding area was defined tattoo. During the analysis it became clear that the black ink and area of 0.225 cm2 was analysed at 40-fold magnifica- interfered with the detectability of the stained hairs. There- tion. Twelve men were treated with finasteride, whereas fore, in future studies we will use red ink for the tattoo.
EJD n° 4, vol. 11, July-August 2001 Figures 3-6. The intra-classcorrelation of three differentmeasurements in 10 volun-teers (subjects) from thesame investigator is shownfor hair counts (Fig. 3) andfor cumulative hair thick-ness (Fig. 4). The intra-class correlation of onemeasurement in 5 volunteers(subjects) from two differentinvestigators is shown forhair counts (Fig. 5) and forcumulative hair thickness(Fig. 6).
For both variables the differences between the results In 11 volunteers without AGA (controls) we observed no after 3 months (6 months) and the baseline were calcula- significant difference in the cumulative hair thickness ted. These differences were analysed using a one-sample within the observation time of 6 months (Fig. 8), whereas untreated men showed a continuous and significant In controls and untreated men we noticed no significant decrease in the overall thickness of hairs 3 and 6 months difference in the number of hairs within the observation after the initial visit (Fig. 8 and Table I). By contrast, men time of 6 months. By contrast, men treated with finasteride treated with finasteride showed a continuous and signifi- showed a continuous increase at 3 months (p = 0.055) and cant increase in the number of hairs within the analysed at 6 months (p = 0.021) in the number of hairs within the area (Table I) after 3 (p = 0.034) and 6 months (p = 0.006) analysed area (Fig. 7 and Table I). (Fig. 8), compared to the values obtained at baseline.
EJD n° 4, vol. 11, July-August 2001 Figures 7 and 8. Hair counts and cumulative hair thickness were analysed for 6 months in 11 volunteers without AGA, in5 untreated men with AGA, and in 12 men treated with finasteride (1 mg/day). In controls and untreated men we noticed nosignificant difference in the number of hairs within the observation time of 6 months (between the values at baseline and after6 months). In contrast those men treated with finasteride showed a continuous increase (mean with 95% confidence interval) at3 months (p = 0.055) and at 6 months (p = 0.021) in the number of hairs within the analysed area compared to the values atbaseline. Untreated men showed a continuous and significant decrease in the overall thickness of hairs 3 and 6 months after theinitial visit (baseline). In contrast those men treated with finasteride showed, in comparison with the baseline visit, a continuousand significant increase in the number of hairs within the analysed area after 3 (p = 0.034) and 6 months (p = 0.006). Analysis of anagen/telogen ratio and hair growth rate at Discussion
the vertex and the occiput in volunteers with AGAThe analysed variables were the portion of anagen hairs Numerous hair diseases such as scarring alopecias, alopecia (growing hairs) and the hair growth rate (difference of the areata or trichotillomania, usually do not need a quantita- length of anagen hairs minus the length of telogen hairs tive method to evaluate the amount of hair shedding.
divided by the time of measurement after clipping. The Androgenetic Affluvium, however, the most common form telogen hairs are defined as non-growing hairs. Telogen of hair loss, is typically difficult to quantify and at present and catagen hairs cannot be differentiated).
simple but reliable procedures have not been developed.
For both variables an ANOVA was calculated with the Although scalp biopsies can be justified in that microscopic fixed factors diagnosis (AGA/control) and investigator examination of scalp skin affected by AGA can identify (investigator 1/investigator 2) and the random factor loca- and quantify any changes resulting from treatment, this tion nested under the diagnosis. The p-values were given invasive technique is often not suitable to monitor patients for the two-sided problem. Figure 9 shows the original over a prolonged period of time. The classical trichogram is values of the portion of anagen hairs for the different dia- harmless to the patient and easy to use but not reliable.
gnosis and investigators with 95% confidence intervals of AGA can be defined as an androgen-dependent process in the means. The same is shown in Figure 10 for the length genetically predisposed individuals, where balding is due difference between anagen and telogen hairs. to the continuous miniaturization of affected hair follicles, The model of the portion of anagen hairs explains 96% of changing large terminal HF into small vellus-like hairs [3, the variance, the hair growth 85%. Both models are highly 18, 19]. Any successful treatment should therefore stop or significant. Table II shows the model and the single reverse the process of HF-miniaturization and increase the effects. For both variables the diagnosis is highly signifi- number of terminal HF whilst reducing vellus hair counts.
cant. These results show that an AGA-affected scalp This concept is illustrated by the phase III studies of men reveals a decreased number of anagen hair follicles with AGA treated with finasteride . In these studies (Fig. 9), and these hair follicles grow more slowly macrophotographs were taken and hairs were counted. This (Fig. 10), compared to the hair follicles at the occiput.
technique produces counts of “visible” hairs, which meansthat tiny vellus-like hairs cannot be seen or counted.
However, during treatment, these vellus-like HF get biggerand subsequently increase the hair count results when themacrophotograph method is used. A major disadvantage of this technique is that it cannot monitor the expected conti-nuous increase in hair thickness during treatment. As a consequence the phase III studies of men with AGA trea-ted with finasteride revealed that the increase in hair counts reaches a plateau after one year of treatment, whe- reas the hair coverage analyzed by global photographs increased continuously . This increase in hair coverage is due to an increase in hair thickness as shown by histolo- gical examination , the direct measurement of hair thickness  and by the continuous increase in hair weight . Although the Ludwig pattern of AGA in EJD n° 4, vol. 11, July-August 2001 Figures 9 and 10. While defining anagen hairs as growing and telogen hairs as non-growing hairs three days after shaving, theTrichoScan is able to calculate a digital trichogram. This figure illustrates the results (mean with 95% confidence interval) fromtwo investigators, who analysed of 18 volunteers with AGA the proportion of anagen hairs (Fig. 9) and the hair growth rate(Fig. 10) at the vertex and at the occiput (only 14 images). Compared to the occiput, the AGA-affected scalp reveals a decreasednumber of and slower growing anagen hairs. Both investigators produced similar results. women differs in appearance  from the Hamilton pat- by coloring the hairs prior to taking the images, without tern occurring in men, these pathophysiological mecha- any negative effect on the collected data. Furthermore, we nims seem to be the same, because female AGA-patients have created an entirely automatic software for the analysis treated with cyproterone acetate [24-26] or minoxidil [27, of the aforementioned parameters of hair growth. Because 28] experience an increase in hair thickness [24-26, 29] or the described technique is a modified and computerized tri- hair weight . Therefore, a reliable hair counting chogram we called it TrichoScan. The images were taken method should primarily be able to calculate the number with a video system for epiluminescence microscopy and thickness of hairs, which is stable within at least 1cm (ELM). ELM is a standard procedure for the analysis of above the scalp [30, 31], in a defined area of the scalp.
melanocytic nevi [35-38], and many dermatologists in From a clinical perspective the hair growth rate (mm/day) Europe already use ELM-systems in daily clinical practice.
and the anagen/telogen ratio are of secondary importance.
These devices produce high quality and reproducible digital As early as 1964 Barman et al.  related a method that images, because the images are always taken at the same used optical contact microscopy to calculate these parame- distance of the lens to the skin surfaces. Our results suggest ters, and much later Hayashi et al.  described a similar that ELM-systems can be used for the evaluation of approach for the measurement of hair growth by the use of patients complaining from androgenetic effluvium and for optical microscopy and computer analysis. However, these authors were unable to automate the process of calculation Variations that normally occur in hair length, weight, and measured the thickness of hairs visually with the cursor thickness, etc., can be assayed either with reference to on the computer monitor. The authors calculated that the standardized values, or by comparing measurements made results from different investigators, but from the same image, on two or more occasions over a given period of time.
differ by ± 8.4%, which makes such semiautomatic methods Similar means of assay must be employed to study as this unsuitable for clinical practice. A nearly similar changes in hair growth which may occur with regard to approach has been tested with the use of the phototrichogram age or illness. The margin of error of the techniques and (PT). The PT has proven to be a suitable and non-invasive the instruments employed should be smaller than the tool to monitor the hair growth phases in situ. This technique magnitude of the variations to be measured. As our results has been improved by the image analysis  and later with show, the TrichoScan fulfils these criteria and has advan- the use of immersion oil and digital contrast enhancement tages over standard procedures used so far for hair measu- . However, although a marked improvement of the rements. Firstly, it is investigator independent. In other images and more accurate quantitative data were noted,  studies using the unit area trichogram, a substantial diffe- rence between the collected data from different investiga- Until now the analysis of the images has been a tedious tors was noted. In these studies a significantly larger mean and time consuming process. Attempts to automate the pro- total hair count was reported from experienced versus cess have been performed several times unsuccessfully [33, inexperienced observers . Our results show, that this is 34, 39]. This is mainly because the HF on the scalp grow not the case for the TrichoScan technique. Secondly, many in groups (follicular units) rather than singly and therefore methods are not strictly validated. The hair weight test is a neighboring HF typically overlap or may be aligned in good example where the hair is clipped in a defined target parallel. Furthermore, any photographic analysis software area. However, the sample error for different investigators needs good contrast between the HF and the scalp skin to is unknown. This is mainly due to the methodology itself, be analyzed, and the fact that many hairs lose their natural because once the hairs are clipped a second investigator pigmentation due to aging or AGA, makes them much cannot clip the same area again to assess the reproducibi- more difficult to detect. We have overcome this difficulty lity of the method. In contrast the TrichoScan is highly EJD n° 4, vol. 11, July-August 2001 validated with defined values for intra-class correlation 9. Friedel J, Will F, Grosshans E. Phototrichogram: adaptation, standardi-
between the same and different investigators. This is of zation and applications. Ann Dermatol Venereol 1989; 116: 629-36.
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EJD n° 4, vol. 11, July-August 2001
La verdad es una; el error, múltiple. No es casual que la derecha profese el pluralismo. Las doctrinas que la expresan son harto abundantes para que, aquí, se pretenda examinarlas todas con seriedad. Pero los pensadores burgueses, que prohíben a sus adversarios utilizar los métodos de Marx si no aceptan en bloque todo el sistema de éste, no vacilan en mezclar con escepticismo ideas tomadas