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Biopsies
for diagnosing hair loss
On the outside, one form of hair loss can look much like another
form of hair loss. There are are basically three hair loss presentations;
diffuse hair loss, patterned hair loss, and isolated patches of
hair loss. Patterned hair loss has just one cause - androgen action
on androgen responsive hair follicles. But diffuse hair loss could
also be androgen induced, a genetic defect, or a telogen effluvium
due to a variety of external or internal causes. Patchy hair loss
could be the result of various localized skin infections, alopecia
areata, a congenital defect, or a scarring alopecia. So on the outside,
one form of hair loss can look much like another, but have a very
different underlying cause. To help differentiate between different
disease diagnoses, the dermatologist has to resort to using various
tests. Blood can be tested in many ways depending on the suspected
problem underlying hair loss. But sometimes blood tests are not
appropriate or reveal nothing abnormal - what then?
The dermatologist might decide to look directly at the subject
of the problem - the hair follicles. To do this a dermatologist
will take a "biopsy". A biopsy is just a small piece of
skin. Depending on the nature of the problem the biopsy will vary
in size, but usually for hair loss conditions a biopsy is almost
never more than 8 millimeters in diameter and is often smaller.
A two millimeter diameter biopsy is not unheard of. The biopsy is
processed so that the dermatologist or a pathologist can look at
the hair follicles under a microscope. The changes in the nature
of the hair follicles and the skin around them can say a lot about
the cause of any hair loss. So when the clinical observations are
not distinctive for a disease and blood tests are irrelevant, a
biopsy can sometimes be the only approach left to finding out what
exactly is going on in an individual with hair loss.
However, many dermatologists are reluctant to take a biopsy. Partly
this may be because the patient does not like the idea of having
one. But there are also questions of the dermatologist's personal
preference and their experience with biopsies. Some dermatologists
come from a school of thought that regards a biopsy as a time consuming
process that provides limited added information. Other dermatologists
regard a biopsy as extremely useful and will readily take biopsies
from their patients. The difference probably depends on which dermatology
department they trained in and the preference of the dermatologists
there. The personal preferences of dermatologists are often handed
down from teacher to student. Having lived and worked in the USA
and Europe, it seems to me that in general a European dermatologist
is more inclined to take a biopsy to diagnose a hair loss condition
compared to a US dermatologist. The chances of a biopsy being taken
will also depend on the dermatologist's level of experience with
biopsies. If the dermatologist has little experience in observing
a biopsy and interpreting it (a process called pathology), then
they will be most reluctant to take biopsies. Unfortunately today
a lack of experience in pathology is all too common among many doctors,
it seems to be a rather neglected area of training.
Biopsy
method and processing
Taking a biopsy is a straight forward procedure that should take
no more than 20 minutes and usually takes less than 10. The main
problem is in deciding what part of the skin to take the biopsy
from. There is something of an art to picking the right place to
take a biopsy that will provide the most information. For example,
the logical place to take a biopsy from a spot of hair loss would
be the center. However, the best place to take a biopsy with spotty
hair loss is often from the advancing edge of the hair loss. In
this location the hair follicles should in the active process of
what ever the hair loss condition is. The hair follicles at the
center of the hair loss spot may be so damaged that they could be
difficult to interpret. So if you have a biopsy done you might find
the dermatologists takes the tissue sample from an area of skin
next to the affected area rather than actually in the affected area.
The area of skin where the condition is will be prepared. If there
is still some hair in the area to be biopsied this will be clipped
away and surrounding hair combed out of the area and held in place
with hair clips. The spot from which the biopsy will be taken is
wiped with a sterile gauze pad soaked in antiseptic. The dermatologist
will then give a local anesthetic by injection into the area to
be biopsied. After a few minutes the dermatologist will take a small,
sterile, cylindrical shaped "punch", and press it into
the prepared skin area. The punch has a sharp razor edge to it so
it cuts a small hole in the skin. Punches come in different diameter
sizes so the dermatologist will choose the right sized punch for
the job. Occasionally, if a larger area of skin needs to be taken,
the dermatologist will use a scalpel to remove the skin instead.
Once the outline of the biopsy has been cut, the dermatologist will
take forceps and a pair of surgical scissors to pull the biopsy
specimen up and cut underneath with the scissors to remove the biopsy.
The biopsy is put to one side. If the biopsy site is very small,
then a sterile gauze pad may be applied to the wound until it stops
bleeding after a couple of minutes. If the biopsy site is bigger
the dermatologist may put in a stitch to close up the wound.
There are two basic things that can be done with a skin biopsy,
it can be "fixed" or it can be "frozen". Usually
skin biopsies will be fixed. This involves placing them in a solution
that promotes a chemical reaction in the tissue that results in
the tissue becoming "fixed" in place and much less able
to biodegrade. There are various solutions that can be used and
each have different fixation properties, but the most common is
a neutral buffered 10% formalin solution. The Formalin is a called
a fixative. It works by crosslinking amino groups in the tissue
and holding them in place. However, sometimes this presents a problem
for analysis later on if studies are to be done to look at specific
protein antigens in the hair follicles. If this is the main aim
then the tissue will be embedded in a wax mixture, dipped into liquid
nitrogen, and then stored until use in a deep freeze. This process
snap freezes the tissue and stops biodegradation but ensures that
the protein antigens survive and are not altered by any fixation.
Freezing a biopsy sample is most likely to be done for scientific
research or very detailed investigations while fixation is the much
more common approach done for routine screening.
Once the tissue biopsy is fixed or frozen it can be stored like
this for some time without worry that the sample will degrade or
change in some way that would influence the interpretation of the
biopsy on examination. When ready, a lab technician will process
the tissue so that it can be examined under a microscope. With fixed
tissue, the biopsy is processed by dipping it in various alcohols
and oils and eventually embedding it in hot wax. The wax is allowed
to set and this helps to physically hold the tissue in place making
it easier to cut. Once set, the wax block containing the tissue
is attached to a "microtome". A microtome is a machine
which contains a sharp knife that can cut very thin slivers of wax
and tissue. Usually the technician cuts between 4 and 6 micrometer
thick slices. These slices of wax and tissue are laid out on glass
microscope slides. Because the slices are so thin they often adhere
themselves to the glass slide without any help, but sometimes the
glass slides are washed in the gelatin solution first and this makes
them sticky so the tissue slices are glued to the slide. Once in
place on the glass slide, the tissue slices are dipped in special
oils to dissolves the wax away, then in alcohols and then in special
dye stains. Depending on what the dermatologist/pathologist wants
to look at, the tissue slices will be stained with different dyes.
Often, consecutive slices of tissue will be stained in different
ways so that when the slices are examined the dermatologist/pathologist
can identify different features in the same part of the biopsy.
With frozen tissue samples, the tissue biopsy, embedded in special
mixture will be attached to a freezing microtome called a "cryomicrotome".
This is basically a microtome in a freezer. The cold keeps the block
and tissue frozen rock solid so it is easy to cut into thin slices.
These slices are also laid out on glass slides and can be processed
in various ways. The tissue slices can be processed by dipping them
in dye stains as with fixed tissue, by more usually frozen tissue
goes through a very complex process called "immunohistology".
With immunohistology a dermatologist/pathologist is exploiting the
fact that antibody proteins that our immune systems produced naturally
against pathogens like bacteria can also be made artificially in
a laboratory. Antibodies are little pieces of protein that will
bind to protein antigens if they come into contact with them. Antibodies
float around in our blood until they come across, for example, a
bacteria. The antibodies bind to the proteins on the surface of
the bacteria and can destroy it through several different methods.
See the immunology section of keratin.com if you want to find out
more about antibodies.
Scientists can make these antibodies in a laboratory and they
can be made to target any protein of interest. So for example, in
alopecia areata research dermatologists are very interested in lymphocyte
cells and what they do inside hair follicles. In the laboratory,
scientists can make antibody proteins that specifically bind to
a particular type of lymphocyte (there are many types). These purified
antibody proteins are widely available from many companies around
the world. If a dermatologist wants to look at a particular lymphocyte
he/she buys a solution of purified antibodies that will bind to
that particular type of lymphocyte. This antibody in a saline solution
is applied on top of the tissue biopsy slices on the glass slide.
The antibodies will only bind to the lymphocytes they are supposed
to target if there are lymphocytes there (if the right protein antigens
are there). Where the antibodies bind to the tissue indicates a
lymphocyte of interest is there in that location. The antibodies
can then be stained to show where they are and this in turn shows
where the lymphocytes of interest are in the tissue specimen. Immunohistology
can be done to find many cell types and also non-cellular proteins.
In this way a very detailed picture can be built up about the nature
of the tissue biopsy and things that are different compared to normal
skin. Immunohistology is mostly done for research purposes, but
occasionally the process may be done for routine diagnosis.
Once the tissue slices have been processed they can be coated with
a clear adhesive and another thin piece of glass stuck on top. This
sandwiches the tissue between glass and permanently holds the tissue
in place. Once this has been done the biopsy samples are fully preserved
and can be stored for hundreds of years without deteriorating.
Reading
a biopsy
The glass slides with the thin slivers of stained tissue in them
are then examined under a microscope. The tissue slices are so thin
that light can pass through them, but because of the stains used
in the processing, different parts of the tissue can be seen as
different colors. This helps to identify different parts of the
tissue and, especially in the case of immunohistological procedures,
individual cells of interest can be picked out by their color. A
pathologist or dermatologist will "read" the slide. That
is, they will look at it and interpret what they see in terms of
what disease may be causing the hair loss.
A normal healthy anagen stage hair follicle that is producing a
hair fiber looks different from a hair follicle that is in a resting
telogen state. So the first thing that may be done is to count how
many telogen hair follicles are in the biopsy slices. In normal
individuals there would not be more than 20% of hair follicles in
telogen. If there are more than that then there is a problem - perhaps
telogen effluvium or pattern baldness are involved. Hair follicles
may also "miniaturize" (get smaller). This miniaturization
is most commonly seen in pattern baldness and it is pretty distinctive
for this form of hair loss. There may be inflammation around and
in the hair follicles. Depending on the location of the inflammation,
whether up near the skin surface, or down at the hair root, the
problem could be alopecia areata or a scarring alopecia. Maybe the
inflammation is diffuse all through the skin in which case the problem
might be something like lupus. Maybe there are structural defects
to the hair follicle. Depending on what defect is present, the problem
might be a genetic one. Finally, the hair follicles may not be there
in their usually density. Some or all of the hair follicles may
have been destroyed by the disease process which might suggest the
late, burnt-out stage of a scarring alopecia.
It is not possible to explain all the the things a pathologist/dermatologist
will look for when evaluating a slide in just a paragraph. Many
different aspects of the biopsy composition are looked at. When
the pathologist/dermatologist has read all the slides with their
different stains from a biopsy, he/she will write a pathology report
detailing the features they have seen that are different from normal
and the conclusion they have come to. If the features in the biopsy
are distinctive enough then the pathologist/dermatologist should
be able to state a clear diagnosis.
Biopsy
references
- Sperling LC. An atlas of hair pathology
with clinical correlations. CRC Press-Parthenon, Boca Ranton,
ISBN: 1842142038, 2003.
- Chamberlain AJ, Dawber RP. Methods of evaluating
hair growth. Australas J Dermatol. 2003 Feb; 44(1): 10-8.
- Van Neste MD. Assessment of hair loss:
clinical relevance of hair growth evaluation methods. Clin Exp
Dermatol. 2002 Jul; 27(5): 358-65.
- Chartier MB, Hoss DM, Grant-Kels JM. Approach
to the adult female patient with diffuse nonscarring alopecia.
J Am Acad Dermatol. 2002 Dec; 47(6): 809-18; quiz 818-20.
- de Lacharriere O, Deloche C, Misciali C,
Piraccini BM, Vincenzi C, Bastien P, Tardy I, Bernard BA, Tosti
A. Hair diameter diversity: a clinical sign reflecting the follicle
miniaturization. Arch Dermatol. 2001 May; 137(5): 641-6.
- Sperling LC. Hair and systemic disease.
Dermatol Clin. 2001 Oct; 19(4): 711-26.
- Sperling LC. Scarring alopecia and the
dermatopathologist. J Cutan Pathol. 2001 Aug; 28(7): 333-42.
- Whiting D. Dermatopathology of common hair
problems. J Cutan Med Surg. 1999 Nov; 3 Suppl 3: S3-13.
- Frishberg DP, Sperling LC, Guthrie VM.
Transverse scalp sections: a proposed method for laboratory processing.
J Am Acad Dermatol. 1996 Aug; 35(2 Pt 1): 220-2.
- Lee MS, Kossard S, Wilkinson B, Doyle JA.
Quantification of hair follicle parameters using computer image
analysis: a comparison of androgenetic alopecia with normal scalp
biopsies. Australas J Dermatol. 1995 Aug; 36(3): 143-7.
- Tsuji Y, Ishino A, Hanzawa N, Uzuka M,
Okazaki K, Adachi K, Imamura S. Quantitative evaluations of male
pattern baldness. J Dermatol Sci. 1994 Jul; 7 Suppl: S136-41.
- Whiting DA. Diagnostic and predictive value
of horizontal sections of scalp biopsy specimens in male pattern
androgenetic alopecia. J Am Acad Dermatol. 1993 May; 28(5 Pt 1):
755-63.
- Sperling LC. Hair anatomy for the clinician.
J Am Acad Dermatol. 1991 Jul; 25(1 Pt 1): 1-17.
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