To avoid receiving laconic findings " chronic dermatitis is important to know how to pick the animals from which to get a biopsy to identify lesions best suited to be sent to the lab and make a withdrawal of the art
Directions
Skin biopsy is indicated in the following situations:
- if the lesions have an unusual appearance
- if the disease does not improve despite the treatments that seemed most suitable
- if you suspect an illness that needs therapy for long periods and not be diagnosed by other means (for example, on suspicion of autoimmune disease)
- if you suspect a disease that requires treatment which may be harmful or contraindicated for the animal and is not diagnosed by other means (eg autoimmune disease)
- If you suspect a tumor and you want to identify the nature of the first dell'intevento excision (eg mast cell) or where it is impossible to perform surgical excision (for esempio linfoma epiteliotropo) e l'esame citologico non sia diagnostico
- in caso di alopecia non infiammatoria ove si siano escluse tutte le cause ormonali
- in caso di prurito ove si siano escluse tutte le cause parassitarie, allergiche e infettive
- in caso di sospetto di malattie congenite (ad esempio nevi)
- in caso di difetti di cheratinizzazione (ad esempio adenite sebacea)
- in generale in ogni caso di dubbio diagnostico
Preparazione dell'animale
Se le condizioni dell'animale lo permettono, è preferibile eseguire le biopsie dopo la somministrazione di una o due settimane di antibiotico, in modo da eliminare infezioni batteriche complicanti che potrebbero disturbare la lettura histology. For this purpose we prefer cefadroxil administered 20-30 mg / kg SID, cephalexin 20-30 mg / kg BID or amoxicillin and clavulanic acid 20-25 mg / kg BID. Antibiotic therapy should be continued until one week after withdrawal of the biopsy, so as to prevent it from infecting the wounds and scars from forming too obvious. If the animal has been subjected to steroid therapy, provided that the patient's condition allow to postpone the withdrawal of the biopsy, it is best to wait 15-20 days after discontinuation of steroid therapy.
Preparation of the field
To collect skin biopsies is important not to make a radical hair removal and disinfection surgery to avoid di rimuovere gli strati più superficiali dell'epidermide che sono spesso importanti per la diagnosi (croste, pustole, squame). E' importante tagliare il pelo con una forbice da pelo, ad una lunghezza di 0,5 cm, facendo attenzione di lasciare le lesioni intatte. La presenza di peli troppo lunghi dà fastidio al momento dell'esecuzione del prelievo e della lavorazione della biopsia, mentre se il pelo viene tagliato troppo corto (meno di 5mm) risulta poi difficoltoso per il laboratorio orientare la biopsia per la lavorazione istologica. E' sufficiente poi spruzzare un poco di disinfettante incolore sulle lesioni da prelevare e lasciarlo evaporare senza tamponare la superficie cutanea. Se si esegue il prelievo in anestesia locale si marcano le lesioni che si desiderano prelevare con un cerchio di pennarello indelebile.
Anestesia
Si preferisce in genere l'anestesia locale, dato che il prelievo si esegue velocemente, è di piccole dimensioni e necessita solo uno o due punti di sutura. Nel sottocute sotto la lesione da prelevare (marcata preventivamente con un cerchio di pennarello indelebile) si iniettano da 0,5 a 1 ml di lidocaina 2% (lidocaina all'1% per i gatti, massimo 2 ml/gatto) o anestetico locale analogo, distribuendolo in diverse direzioni. Prima di eseguire il prelievo è consigliabile aspettare qualche minuto, in cui si possono preparare gli strumenti necessari per il prelievo.
E' però indispensabile l'anestesia generale nei seguenti casi:
- cats, unless they are very quiet and that the levy is made from the trunk. In cats it is possible to inject a maximum of 2 ml of 1% lidocaine diluted with saline to cat, which limits the possibility of taking samples 1-4 only.
- withdrawals from the legs below the elbow joint and knee
- drawings on pads and nail beds
- withdrawals from the head generally (nose, lips, eyelids, pavilions)
- drawings on the genitals, scrotum, anus
- restless or aggressive animals
- lesions in the skin, where it is impossible to run a local anesthetic
selection of lesions
The choice of the lesions sample size is crucial for obtaining an accurate diagnosis. Should preferably be taken more samples (at least 3) from primary lesions or recent appearance, such as macules, papules, pustules, blisters, blisters, nodules and tumors. Should not be sampled from areas with minor injuries, such as punctuation autotraumatismo, local treatments (creams), infection and necrosis. In the case of multiple lesions (or in different stages of development) is good to take multiple biopsies are representative of all types of visible lesions on the animal. It 'should also send a sample taken from an area adjacent to the apparently normal area damaged.
in nodular lesions and deep biopsy should be charged in depth (preferably by excision) in order to obtain a representative sample of the skin.
Picking with punch
Whenever possible we recommend using punch large (6 or 8 mm), to facilitate the work of the laboratory technician and have a greater chance that the biopsy is diagnostic. You can use punch of 8 mm on the trunk and thighs, while the punch of 6 mm should be reserved for the pads and the truffle dogs of medium and large size. The punch 4 mm should be reserved for lesions in specific areas, such as the nose and pads of cats and small dogs and ears (especially the inner surface, where the dermis is not mobile on the cartilage).
is recommended, if possible, to take more samples (minimum 3), especially if the lesions have different appearances, such as a papule, a pustule, a collarette and a macula. It 'also preferable to store samples of different lesions in different containers and numbered separately and describe the appearance of lesions and their location (with respect to the number of container) on the anamnesis form.
The punch should be placed perpendicular to the skin surface, which is stretched between thumb and index finger under the instrument. The lesion to be taken must be placed exactly in the center of the punch, as in the laboratory, biopsy is always cut in half along the diameter of the dowel through the skin. It exerts a continuous pressure on the punch by turning the tool in one direction only, until it has completely punctured the skin and reached the subcutaneous tissue. Now the punch retracts and lifts the plug by grasping the subcutaneous tissue and deep dermis with tweezers and is removed by cutting the stalk of adipose tissue. It 's important not grasp the epidermis and superficial dermis with tweezers to avoid artifacts that might affect the reading of the section.
biopsies taken from skin areas where the subcutaneous tissue is thin or absent (ear) from the underlying tissues are gently unstuck trying to preserve as much as possible the structure of the epidermis. Some areas
skin requires more attention because of the risk of damaging the underlying tissues. These are the phalanges (presence of flexor and extensor tendons), lips (presence of arteries that run along the entire perimeter of the mouth), the pavilion (the presence of cartilage below), truffles (the presence of cartilage below), and where it Note the presence of visible blood vessels (veins breast abdomen, cephalic arm, etc.) that should be avoided. In these areas you should proceed carefully and slowly run shallower samples, if possible by raising a fold of skin between two fingers to remove the skin from underlying structures.
biopsies would come gently blotted on a gauze to remove excess blood, which disturbs the reading histological, and immediately placed in 10% buffered formalin.
hole biopsy is sutured with one or two points depending on the size and location. Exceptions biopsies of the ear (especially those of the inner surface), where, due to reduced mobility of the dermis below the cartilage, it is impossible to bring the wound edges without distorting the pavilion. In these cases it is sufficient to apply a gauze pad over the wound and wrap the ear until you get strettamete spontaneous hemostasis. The biopsy hole is left to heal by secondary intention (usually running 4 mm hole).
Drawing excision
Sampling with scalpel excision is indicated for the following cases:
- pustular lesions or blisters that break easily or very large (larger than the diameter of the punch), which would break during drawing with punch.
- very large flat lesions such as erosions or ulcers. Since the diagnosis is often important to assess the margins of the lesion, samples with the punch, round shape, would be difficult to place in order to be included with certainty in the margins of the lesion. Excisional biopsies taken with the long side perpendicular to the margin of the lesion, as are conventionally cut along the longitudinal axis, are obtained by force sections containing the transition areas of interest for the diagnosis.
- nodular lesions that can be so easily excised in toto.
The sample for diamond excision is performed with a scalpel to include the margin of the lesion or a lesion extended to the center of the biopsy.
Establishment
Both biopsies taken for excision and for those with the punch is important to dry the blood on the surface using absorbent paper and then adhere to the subcutaneous tissue of the biopsy on a piece of wood (spoon down language) or on a card , and put everything in formalin. This will prevent the biopsy is enriched and greatly facilitates the work of the pathologist cutter. The lesions small to be taken in its entirety (with punch or excision) of the big ones you should take a sample of the margins, at the turn of the sick and the healthy tissue (excision).
Because formalin fixation with the colors disappear and the texture becomes smooth and solid lesions that are very evident in fresh (erythema, palpable nodules, etc.) may not be so when the fabric is attached. In this case, then it is advisable to mark the specific lesion with sutures and attach a detailed macroscopic description. For the same reason it is important to the levy so that the lesion is located perfectly in the center, because conventions for all biopsies are in fact cut perfectly in half
Sample and base are then either immediately placed in formalin, making sure to submerge the sample well and do not float on the surface. For nodules larger than 2 cm in diameter is important to make cuts at full thickness to 1-2 cm apart, leaving the pieces joined at the base, like a loaf of bread, so to guide the pathologist on the shape and size of the piece. For parts of a larger than normal histological skin biopsy is important to respect the volume ratio of 1:10 between the tissue to be fixed and formalin. You should also send the pieces in larger containers with the opening of the piece itself, because formalin hardens the tissues, then making it impossible to extract container from the mouth narrower than their diameter.
Formalin removes the bound water to the tissues and cause hardening. In this way, the tissues become resistant to hydrolytic enzymes. Formalin is not perfectly stable, and in contact with oxygen turns into formic acid. The latter is not well fixed tissues and causes artifacts that disturb the histological examination. For this reason we recommend that you use buffered formalin at pH 7 and to throw more than one year old. Due to the high toxicity of formalin is recommended to not touch it with bare hands and throw it with the private waste speciali.Appena of their blood supply, the tissues begin a process of autolysis, which causes destruction of cells. The proliferation of saprophytic bacteria causes the degeneration of tissues. It 'important to put each sample in formalin immediately after collection and not expect to have collected all the samples before fastening. A small skin biopsy can show up to essiccarisi microscopically visible artifacts, even if only 5 minutes left in the heat id an operating light. The minimum period of fixation in formalin for 24 hours.
Enter the laboratory before being sent to the laboratory, samples should not be kept in the refrigerator, as if frozen by accident would be obtained artifacts in the sections. For this reason, if sent by courier in the winter is particularly cold climate areas (if there is a risk that the temperature falls below zero) is to add alcohol to a 10% formalin, to prevent freezing.
samples should always be accompanied by a comprehensive medical history form. It 's very important to remember that the more detailed the information you give to the pathologist, the more likely it is that you receive an accurate diagnosis or useful. It 'better to put samples from different lesions or different sites in different containers and numbered on the application form and specify the source or type of injury for each sample.
A thorough compilation of the application form in its entirety helps a lot in the formulation of the dermatopathologist diagnsi more accurate. In particular, it is important to provide data on the species, breed, age and sex of the animal, the distribution of lesions and the site of collection of biopsies. The macroscopic description of lesions is essential, as well as information on the duration of the disease and the possible presence of systemic manifestations, including pruritus.
Finally it is also important to provide information on the therapies administered, the type of drug, the duration of treatment, its effect and the last administration. A list of differential diagnoses can be completed the anamnesis.
What happens in the laboratory?
biopsy is removed from formalin and was sectioned into two halves along the longitudinal direction of hair growth. For this reason it is important to leave some 'hair to guide the cutter. Then the samples are processed with alcohol and xylene and placed in a block of formalin. The block is then passed to the microtome which cut into thick slices 4 to 6 um. These sections are then collected on a glass slide and stained as necessary. The colored samples are then mounted with a coverslip and a drop of glue. So I'm ready to be read by dermatopathologist. It 'important to choose a veterinarian with laboratory a pathologist with special interest in dermatopathology. It 'absolutely not advisable to consult a pathologist or dermatopathologist in human medicine. Finally it should be remembered that the time technicians are working on a piece of at least two days, not including the reading of the pathologist, but can stretch to a week or more if you need the decalcificaizione, special stains ol'immunoistochimica.
What to expect from a biopsy report?
Sometimes, despite being carried out testing of the art, the biopsy report does not provide a definitive diagnosis, but it just gives some general tips. This happens because some aspects are common histopathological more dermatoses (eg. endocrinopathies or the allergies) and since the veterinary clinical biopsies levying especially unusual cases, disorders of which little is known about the etiology or pathogenesis. A negative response ("no ...") is also has its value and should be given careful consideration. Finally, we must always remember that the skin biopsy revealed the situation at the time of collection, and that any skin disease on the other hand has a development that may change over time. Make a diagnosis with a still image of a dynamic process can sometimes be very difficult. It is the task of the clinician to assess the histopathological report in the light of the clinical manifestations of the animal and choose la terapia più appropriata.
Bibliografia
1 Angarano DW. Biopsies of the skin and mucous membranes. Seminars Vet Med and Surg (Small Anim) 8(4):235-8, 1993
2 Dunstan RW. A user's guide to veterinary surgical pathology laboratories or, why do I still get a diagnosis of chronic dermatitis even when I take a perfect biopsy? Vet Clin North Am Small Anim Pract 20:1397, 1990
3 Longeart L, George C, Le Net JL. L'examen histopatologique: la biopsie cutanée. Le Point Vétérinaire 26(162):71-2, 1995
4 Mechelli L, Floridi C, Fondati A. La biopsia cutanea nella diagnosi dermatologica veterinaria. Veterinaria 5(1):93-6, 1991
5 Yager JA, Wilcock BP. Skin biopsy: revelations and limitations. Can Vet J 29:969,1988
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