**3. Trichogram**

In this procedure, 60–80 hairs are plucked with a rubber‐armed forceps from a 5‐day unwashed hair. Hair bulbs are immediately placed with their roots on a glass slide in an embedding medium, which allows information about the state of the proximal end of the hair shaft (the root), the distal end (the tip) and hair root is necessary [98].

The trichogram is a useful complementary tool for clinical evaluation, diagnosis, and the monitoring of treatment response [99].

It should be noted that the trichogram simply provides a snapshot of the hair follicle at the time of examination and that the condition of follicles can vary within the same patient depending on numerous factors, such as sampling site, previous washing or brushing of the hair, and time of the year [100].

#### **3.1. Trichogram equipment**

Appropriate sampling site for male pattern hair loss should be taken from the central interparietal area, while the second sample, if needed, should be taken from the temporal or occipital area. In female pattern hair loss, samples should be taken from the center and the vertex of the scalp. The sites for telogen hair loss and scarring alopecia are, respectively, the central interparietal area and the advancing border of the alopecic patch. Using a rubber‐sheathed Kocher forceps, a tuft of 15–20 hairs must be removed. To do this, you have to place the forceps 1–2 cm from the scalp and pluck out the hairs rapidly and firmly in the direction of the natural growth of the hair. If the hairs are not plucked out firmly, they may appear as pseudo‐ dystrophic hairs under the microscope, or exhibit frayed or broken roots [99].

The next step is to prepare the hairs for examination under the microscope. They should be parallel to each other and that the roots are aligned. Next, they are covered with clear adhesive tape. To avoid artifacts and obtain a sharper, cleaner image, you may apply several drops of balsam (such as that used to mount histological slides) and cover the hairs with a cover slip. The use of polarized light improves image quality [99].

#### **3.2. Hair examination**

The sample is examined using a 4× objective, although a 10× or 40× objective can be used if higher magnification is needed. A higher‐quality image can be obtained by fitting 2 polarizers to the microscope: 1 between the condenser and the sample and the other between the sample and the observer [99].

#### *3.2.1. The proximal end*

Anagen hair shafts are longer, have a uniform diameter, a rectangular shape, and a slight distal angle. Pigmentation is intense in the bulb area and there are sheaths and membranes.

Telogen hair shafts are shorter and appear higher up in the trichogram, above the roots of anagen hairs; the root is thick and club shaped and there are no distal angles.

Pigmentation is weak or absent; the sheath is also absent or found only at the distal end.

Very few hairs in the catagen phase are observed in the trichogram as they account for a very small percentage of all hair.

The anagen to telogen ratio varies, mainly according to age and sex. Children have the highest percentage of anagen hair (95% anagen vs. 5% telogen), and the ratio decreases with age. The anagen to telogen ratio is 86:11 in women and 83:15 in men. In a normal trichogram, an average of 89% of hairs are in anagen, 10% in telogen, and 1% in catagen. A diagnosis of telogen effluvium is established when over 20% of the hairs examined are in telogen phase.

Dystrophic hairs have a decreased proximal diameter, an irregular contour, no epithelial sheaths, and an angle of over 20. They are common in AGA or in hair that has not been removed correctly from the scalp. Keratotic material may be observed on the tip of the hair in conditions such as seborrheic dermatitis, psoriasis, and folliculitis. A common finding in patients with demodicosis is the presence of Demodicosis folliculorum in contact with the root of the hair, although this condition is usually diagnosed by superficial skin biopsy [99].

#### *3.2.2. The hair shaft*

Normal hair is uniform in appearance and structure along the entire length of the hair shaft; this uniformity is also observed between the different hairs in a sample. Hair dysplasias are malformations of the hair shaft. Although scanning electron microscopy is the diagnostic tool of choice in such cases, certain signs may be observed in the trichogram [101]:

#### *3.2.2.1. Monilethrix (beaded hair)*

Alternating segments of narrowings and nodosities, giving a characteristic beaded appearance.

#### *3.2.2.2. Pseudomonilethrix*

Round hairs with irregular, sporadic rounded nodosities. There are no narrowings.

### *3.2.2.3. Pili torti*

the scalp. The sites for telogen hair loss and scarring alopecia are, respectively, the central interparietal area and the advancing border of the alopecic patch. Using a rubber‐sheathed Kocher forceps, a tuft of 15–20 hairs must be removed. To do this, you have to place the forceps 1–2 cm from the scalp and pluck out the hairs rapidly and firmly in the direction of the natural growth of the hair. If the hairs are not plucked out firmly, they may appear as pseudo‐

The next step is to prepare the hairs for examination under the microscope. They should be parallel to each other and that the roots are aligned. Next, they are covered with clear adhesive tape. To avoid artifacts and obtain a sharper, cleaner image, you may apply several drops of balsam (such as that used to mount histological slides) and cover the hairs with a cover slip.

The sample is examined using a 4× objective, although a 10× or 40× objective can be used if higher magnification is needed. A higher‐quality image can be obtained by fitting 2 polarizers to the microscope: 1 between the condenser and the sample and the other between the sample

Anagen hair shafts are longer, have a uniform diameter, a rectangular shape, and a slight distal angle. Pigmentation is intense in the bulb area and there are sheaths and membranes. Telogen hair shafts are shorter and appear higher up in the trichogram, above the roots of

Pigmentation is weak or absent; the sheath is also absent or found only at the distal end.

Very few hairs in the catagen phase are observed in the trichogram as they account for a very

The anagen to telogen ratio varies, mainly according to age and sex. Children have the highest percentage of anagen hair (95% anagen vs. 5% telogen), and the ratio decreases with age. The anagen to telogen ratio is 86:11 in women and 83:15 in men. In a normal trichogram, an average of 89% of hairs are in anagen, 10% in telogen, and 1% in catagen. A diagnosis of telogen effluvium is established when over 20% of the hairs examined are

Dystrophic hairs have a decreased proximal diameter, an irregular contour, no epithelial sheaths, and an angle of over 20. They are common in AGA or in hair that has not been removed correctly from the scalp. Keratotic material may be observed on the tip of the hair in conditions such as seborrheic dermatitis, psoriasis, and folliculitis. A common finding in patients with demodicosis is the presence of Demodicosis folliculorum in contact with the root of the hair, although this condition is usually diagnosed by superficial skin biopsy [99].

Normal hair is uniform in appearance and structure along the entire length of the hair shaft; this uniformity is also observed between the different hairs in a sample. Hair dysplasias are

anagen hairs; the root is thick and club shaped and there are no distal angles.

dystrophic hairs under the microscope, or exhibit frayed or broken roots [99].

The use of polarized light improves image quality [99].

**3.2. Hair examination**

92 Hair and Scalp Disorders

and the observer [99].

*3.2.1. The proximal end*

small percentage of all hair.

in telogen phase.

*3.2.2. The hair shaft*

Twists of hairs with bending at different angles and regular intervals.

#### *3.2.2.4. Trichorrhexis invaginata (bamboo hair)*

Ball‐shaped deformity with cupping at the proximal end of the hair shaft.

#### *3.2.2.5. Trichothiodystrophic hair*

Patients with trichothiodystrophy may have hair with ribbon‐like flattening, characteristic trichoschisis‐like fractures (clean transverse breaks), with an irregular surface, and tiger‐tail banding.

#### *3.2.2.6. Trichonodosis*

Single or double knots on the hair shaft. Tie knots and other more complex knots are also observed.

#### *3.2.2.7. Trichorrhexis nodosa*

Bulging hair characterized by fracture nodes with open splitting of the cortex on both sides of the node. If the hair eventually splits, it will leave a brush‐like appearance at both ends.

#### *3.2.2.8. Bubble hair*

Short, broken hairs with a wavy surface,and bubbles inside the shaft.

#### *3.2.2.9. Loose anagen hair*

Twisted anagen hairs with a ruffled cuticle at the proximal end. Long, pili canaliculi type canals on the shaft are a common finding [102].

#### *3.2.2.10. Pili annulati*

Hair shafts with alternating light and dark bands.

#### *3.2.2.11. Woolly hair*

Thin curly hair forming small woolly balls.

#### *3.2.2.12. Uncombable hair (pili canaliculi)*

Canalicular formation along the entire length of the hair shaft. This formation can be difficult to spot under a microscope but the micrometer can be moved if pili canaliculi is suspected.

#### *3.2.3. The distal end*

Three types of hair shaft tips can be observed:

Javelin tip: A very sharp, spear‐like tip is seen in hair that is growing well and has never been cut, paintbrush tip: Fractures in the hair shaft (trichoschisis) give the tip a paintbrush‐like appearance, seen in hair shaft anomalies such as monilethrix, alopecia areata, or in hair fragility induced by cosmetic products an clean‐cut tip: The distal end has been cut and ends in a perfectly straight line. It is typically seen in hair that has been cut and in cases of trichotillomania [99].

#### **3.3. Common alopecia in trichogram**

In alopecia areata, hair shaft is with alternating narrow and normal sections. Pseudomonilethrix and/or trichoschisis may be observed in some hairs. Hair shaft diameter variability has been demonstrated in women, with larger diameters seen in higher stages of the Ludwig Scale [103]; the differences were minimal at stage I and maximal at stage III. In TE, the hairs are shorter than normal, have a uniform diameter, a rounded proximal end (club‐like appearance) and a lack of pigment and membranes. In anagen effluvium normal anagen hairs that are longer than telogen hairs, pigmented, and having sheaths and membranes whose distal end is angled like a golf club are seen in the trichogram. Nits or lice may be seen in patients with pediculosis capitis [99].
