DR ISRAEL G.M

MICROSCOPIC EXAMINATION

CULTURE

SKIN/SURGICAL BIOPSY

IMMUNOFLOURESCENCE TOOLS

WOOD’S LIGHT EXAMINATION

SKIN SNIP

DIASCOPY

PRICK TEST

HISTOLOGY

ELECTRON MICROSCOPY

PATCH TESTS

DNA PROBES

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Subtle changes in the skin become more

apparent when enlarged.

Hand held

Attached to spectacles

Skin surface microscopy

▫ ‘dermatoscopy’,‘epiluminoscopy’ and

‘epiluminescent microscopy’.

▫ high quality magnifying lens and a powerful

lighting system.

▫ Allows examination of skin structures and

patterns

• INDICATION

• Evaluation of pigmented skin lesions.

• Diagnosis of melanoma.

Finding a scabies mite within a burrow

Locating a splinter

Evaluating nail fold capillaries in;

cutaneous lupus erythematosus

systemic sclerosis

• Symmetry or asymmetry

• Homogeny or heterogeny (structural differences across the lesion)

• Distribution of pigment:

▫ network, pseudonetwork

▫ blotches, dots, globules

▫ cobblestoning, ovoid masses

• Vascular pattern: regular or irregular

• Border of the lesion: fading or sharply cut off

• Presence of ulcers

Ink-spot naevus Benign mole

Blue naevus Basal cell carcinoma

Early melanoma Seborrhoeic keratosis

• Spectrophotometric analysis :

• dermoscopy a step further by using a light beam that penetrates to a depth of 2mm (1000x deeper) beneath the skin surface.

• Light images taken with a digital camera or hand-held scanner are then fed into a computer

• identify and diagnose early stage malignant melanomas

•

used in a number of centres in the UK,

Australia

recently been introduced to New Zealand

(SIAscopy).

SIAscope stands for Spectrophotometric

Intracutaneous Analysis ( Astron Clinica

Limited)

• Light energy is absorbed and remitted by particular target cells with colour (chromophores) in the skin.

• 1. The device emits visible and infrared light

• 2. chromophores in the layers of skin respond to the light differently and send back remitted light

• 3. Melanin absorbs ultra-violet light

• 4. Haemoglobin (red blood cells) absorb infrared light

• 5. Collagen absorbs and remits light across the spectrum corresponding to the size and amount of collagen cells in the deeper layers of skin (papillary dermis).

• .

• 6. Reflected light received by the spectrophotometer is analysed by a computer software program that calculates the quantity of light absorbed at various wavelengths.

• 7. The images created from computer analysis show the presence of melanin, blood or collagen changes in the area examined.

• For example, melanocytes may be shown in the deeper layers of skin, which may be an early indication of melanoma

• To identify parasites esp ochocerciasis

• It is taken from subcutaneous areas(for microfilaria) and bony prominences(nodules which contain adult worms)

• Bony prominences-iliac crests,greatertrochanters,ribs,scalp,coccyx

• SCLREOPUNCH

• Skin snips are placed in normal saline under a cover slip

• After 4hrs,microscopy will show microfilaria wriggling free on the slide

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• Should be taken from lesions that are fresh but well developed

• Not be taken from lesions below the kneel-difficult for the pathologist to interpret

• On the face;▫ temple lateral to the eyebrow (the temporal artery)

▫ the nasolabial fold as it intersects the alae (angular artery)

▫ the supraorbital notch at the medial end of the brow (the supraorbital artery).

• Arteries may be injured by a deep punch biopsy at these sites.

• Punch▫ Most superficial inflammatory and bullous diseases;

benign and malignant tumors except malignant melanoma

• Shave▫ Superficial benign and malignant tumors (e.g.,

seborrheic keratoses, warts, dome-shaped nevi, and non-melanoma malignancies)

• Excision▫ Deep inflammatory diseases (e.g., erythema

nodosum); malignant melanoma

• site is prepared for biopsy with an alcohol pad;

• Asterile technique

• Local anesthesia is induced with 1% lidocaine

with epinephrine.

▫ Epinephrine is avoided for biopsy near the fingertips.

▫ The injection is positioned around and under but not

directly into the lesion.

• The surrounding tissue is supported by

stretching the skin with the thumb and index

finger of the free hand.

• The punch is rotated back and forth between the thumb and forefinger while it is simultaneously pushed vertically into the tissue

• The punch is withdrawn and the cylindric piece of tissue is gently supported with smooth-tipped forceps;

• the specimen is cut deep with scissors to include subcutaneous tissue.

• Preservative

• Bleeding is controlled ; pressure or Monsel'ssolution

• Specimens for fungal M/C/S▫ Scrapings of scale ; leading edge of the rash after the

skin has been cleaned with alcohol.

▫ Skin stripped off with adhesive tape, which is then stuck on a glass slide. Hair pulled out from the roots.

▫ Brushings .

▫ Nail clippings.

▫ Skin biopsy.

▫ swab from a mucosal surface

• Transportation; ▫ sterile container

▫ black paper envelope.

• Potassium hydroxide (KOH) preparation, stained with blue or black ink▫ Unstained wet-mount

▫ Stained dried smear

▫ Histopathology of biopsy with special stains.

• Microscopy can identify a dermatophyte by the presence of:▫ Fungal hyphae (branched filaments) making up a mycelium

▫ Arthrospores (broken-off spores)

▫ Arthroconidia (specialised external spores)

▫ Spores inside a hair (endothrix) or outside a hair (ectothrix).

• Yeast infection:▫ Yeast cells ; dividing by budding

▫ Pseudohyphae (branched filaments similar to those of a dermatophyte) forming a pseudomycelium

PAS stain of

Aspergillus seen

in a skin biopsy

KOH preparation

of microsporum canis

showing hyphae

KOH preparation

of candida

showing pseudohyphae

• Culture▫ identifies organism: e.g. a particular animal

▫ To select the most suitable treatment.

• culture may take several weeks,

• incubated at 25-30ºC.

• Medium ;▫ Sabouraud's dextrose agar containing cycloheximide and

chloramphenicol.

• Negative culture :▫ Not due to fungal infection.

▫ specimen was not collected properly.

▫ Antifungal treatment

▫ delay before the specimen reached the laboratory

▫ incorrect. laboratory procedures

▫ Slowly growing organism .

Culture

Antibodies (histoplasmosis,

coccidioidomycosis)

Antigen (cryptococcosis, aspergillus,

candidosis, histoplasmosis)

• Open patch test - this test is used in testing the plant oleoresins.

• Method:

• Acetone extracts of the plants, weeds or trees are applied on the skin surface. The area is kept dry and the result is noted after 48 hours.

• Provocative patch test - detection of sensitivity of neomycin, penicillin and benzocaine.

• .

• Vapor patch test - this test is applied for volatile substances such as perfumes.

• Apply the vapor or gas to the skin surface under a small glass cup tapped into the skin for 48 hours.

• Mucous membrane patch test - this test is used for local sensitizing agents for the mouth as mouthwashes, nicotine and toothpaste.

• .

• Photo patch test - this test is used for detection of photosensitizing substances such as phenothiazine, sulfa, and photosensitizing plants.

The first appointment - take about half an

hour.

Tiny quantities of 25 to 150 materials in

individual square

plastic or round aluminium chambers are

applied to the upper back.

They are kept in place with special

hypoallergenic adhesive tape

The patches stay in place undisturbed for 48

hours.

Second appointment

48 HOURS later

patches removed.

Back is marked with an indelible black felt

tip

These marks must still be visible at the third

appointment, usually two days later

The back should be checked and if necessary

remarked on several occasions between the

2nd and 3rd appointments

• The dermatologist will complete a record form at the second and third appointments (usually 48 and 96 hour readings).

• The result for each test site is recorded.

• Grading system we use is as follows:

▫ Negative (-)

▫ Irritant reaction (IR)

▫ Equivocal / uncertain (+/-)

▫ Weak positive (+)

▫ Strong positive (++)

▫ Extreme reaction (+++)

• Irritant reactions include ;

▫ sweat rash, follicular pustules

▫ burn-like reactions.

• Uncertain reactions refer to a pink area under the test chamber.

• Weak positives are slightly elevated pink or red plaques.

• Strong positives are papulovesicles’

• extreme reactions are blisters or ulcers.

• The relevance depends on the site and type of dermatitis and the specific allergen.

++ reaction +++ reaction

Notes

• Do not expose back to the sun for 4 wks b/4 patch tests

• Wear old clothing; felt tip pen marks can stain clothes

• Do not swim, rub, or exercise, as the patches may come off

• Keep the back dry, so no baths, showers or unnecessary sweating

• Arrange for someone to remark the test sites with indelible felt tip marker

• Photopatch tests

• Some patients have photopatch tests because their dermatitis develops on skin exposed to the sun

(photosensitivity).

• Two sets of perfumes, antiseptics, plant materials and sunscreens may be applied.

• After removal, one set is exposed to a small dose of UVA not enough to cause a photosensitivity reaction on its own.

Skin prick testing is an allergy test

used to identify allergens responsible for

triggering symptoms in allergic diseases

Atopy is characterised by an overactive

immune

response to environmental factors and has a

strong genetic component.

It usually manifests clinically as one or more

of the characteristic disorders of asthma,

eczema, or hay fever

Eczema

acute urticaria and angioedema

bee and wasp stings; especially if

immunotherapy is being considered.

performed on the inner forearm.

Any number of allergens can be tested, as

few as 3 or 4 or up to about 25 allergens.

overview .

Clean arm with soap and water or alcohol

The forearm is coded with a skin marker pen

corresponding to the number of allergens being

tested.

Marks should be at least 2cm apart.

A drop of allergen solution is placed beside

each mark

A small prick through the drop is made to the

skin using a sterile prick lancet. .

Excess allergen solution is dabbed off with a

tissue

Observe skin reactions – if a reaction occurs

it should do so within 20-30 minutes

Venison blood allergy

• placing the tests too close together (<2 cm apart)

• Causes of false-positive result

• Positive reaction from one test site may affect the result of a neighboring test site

• Irritant reaction

• Causes of false-negative result

• Medications such as antihistamines that block the effect of histamine

• Decreased reactivity of the skin in infants and elderly patients

• Allergen extract too diluted

The term ‘mole mapping’ has been used in

several different ways.

However, it usually refers to a surveillance

programme for those at high risk of

malignant melanoma

This is an ultraviolet lamp with Wood‘s filters, which

produces a wavelength about 3650 Â

Wood‘s lamp may be used to help in the diagnosis of the

following lesions:

Fungal infections:

Tinea capitis caused by Microsporon species gives

bright blue-green fluorescence.

It should be noted that Tricophyton tonsurans and

Tricophyton violeceum do not give fluorescence.

Erythrasma: gives a coral-red fluorescence.

Pityriasis versicolor lesions when examined in a

dark room with Wood‘s light appear as

sharply accentuated lesions.

Bacterial infections:

Pseudomonas pyocyanea gives a yellowish-green

color due to pyocyanin.

The acne bacillus causes a coral fluorescence in

the follicles possibly due to porphyrin

production.

• Wood‘s lamp can be used to determine the depth of melanin in the skin

• . Wood‘s light accentuates contrast between pigmented and non-pigmented skin

• separates hypopigmented from totally non-pigmented areas as in vitilligo and albinism.

• Detection of porphyrins:▫ Porphyrins in urine when examined in dark field by

Wood's light, gives red color or pinkish orange.

▫ Porphyrins in feces

▫ blister fluid in porphyria lesions

▫ the teeth in erythropoietic porphyria

▫ blood protoporphyria give also the same fluorescence.

Erythropietic porphyria can be also diagnosed by

the fluorescence of red cells.

• Tetracycline: deposits in the growing enamel

teeth of children that produces a typical yellow

color.

• Malignant tumors of the skin especially

squamous cell carcinoma gives bright-red

fluorescence.

• Miscellaneous:

▫ Medications

▫ industrial compounds

These tests are indicated in certain diseases

such as syphilis and non-venereal treponemas

mainly Pinta and Bejel.

Liver function tests

Diseases of liver may manifest with internal

and cutaneous manifestations.

Hormonal essay: is an important line in

investigating certain skin diseases especially

those associated with endocrine

dysfunction.

This test shows precipitation of proteins when cooled which redisolves again when heated.Cryoglobulins are not present in normal individuals.Skin diseases that show positive test:

• PurpuraCold sensitivity cyanosisRaynaud‘s diseaseLupus erythematosusLymphgranuloma venerumLeg ulcersCutis marmorata.

May be present with:

▫ Collagen vascular disease, especially SLE.

▫ Chronic liver diseases.

▫ Hashimoto‘s thyroiditis, thymoma, myasthenia

gravis.

▫ Pernicious anemia.

▫ Tuberculosis.

▫ Leprosy.

▫ Diffuse pulmonary fibrosis.

▫ Lymphoma or other malignancy.

▫ Ulcerative colitis.

• Non-specific test.

• A raised ESR is usually due to an increased aggregation of red cells due to an abnormality of plasma proteins

• Notably an increase in plasma fibrinogen

• Associated with the acute or chronic phase reaction.

• Some causes that raise ESR:

• Physiological▫ pregnancy, menstruation, advancing age.

• Infections.

• Inflammatory disorders, e.g. vasculitis.

• Systemic lupus erythematosus (SLE).

• Tissue destruction.

• Malignant neoplasms.

• Paraproteinaemias.

• Polycythaemia.

Atopic disorders, especially asthma and eczema.

Allergy to food or drugs.

Parasitic infestations: worms (intestinal or

systemic), scabies.

Collagen vascular disease, polyarteritis nodosa,

dermatomyositis.

Bullous disorders: dermatitis herpetiformis,

pemphigus, and pemphigoid.

Erythema neonatorum.

Malignancy, especially Hodgkin‘s disease and

eosinophilic leukemia.

Viral infections

exanthemata

infectious mononucleosis.

Bacterial infections

Tuberculosis

syphilis, brucellosis

typhoid.

Infections, e.g. erysipelas, carbuncle.

Inflammatory disorders including pustular or

inflammatory psoriasis, erythroderma, and

pyoderma gangrenosum.

Systemic malignancy (leukemia).

Reaction to systemic steroid therapy

Cytological exam from the floor of a bulla is

used to confirm diagnoses of bullous

diseases.

In most bullous eruption the smear will show

only inflammatory cells.

pemphigus ;numerous acantholytic cells with

large nuclei and condensed cytoplasm .

Herpes simplex, zoster and varicella lesions: the

smear shows large, multi-nucleated and mono-

nucleated giant cells and ballooning

degeneration of the nuclei.

Amniotic fluid and its cells are used for

diagnosing a variety of metabolic diseases,

morphological, biochemical and cytogenic.

Ultrasonography: is a powerful tool for the

detection of central nervous system and

skeletal disorders.

Fetoscopy: this is a technique that involves

the insertion of a fibro-optic endoscope into

the pregnant uterus.

• Amniocentesis:▫ convenient

▫ relatively safe method of obtaining amniotic fluid and its cells for morphological, cytogenetic and biochemical investigations.

▫ performed at 16 weeks of gestation.

• Fetal skin biopsy:▫ Helps in the detection of morphological or immuno-

histochemical abnormalities.

• Enzymatic assay

• DNA Examination:▫ Helps to detect any genetic abnormalities.