Diagnosis of Vesicullo Bullous Lesions-Simplified

Pathological condition affecting the oral cavity is diagnosed accurately for their appropriate management. Knowledge of clinical presentation of these disorders is necessary because as such oral vesiculo bullous lesion gets ruptured and become erosions, ulcerations hence making the diagnosis of vesiculo bullous lesions more difficult. In this article various diagnostic procedures of vesiculo bullous lesions is explained.


INTRODUCTION
Vesiculo bullous lesions are a distinct group of oral disorders characterized by the formation of vesicle or bullae.And it is uncommon to see vesicle and bullae intra orally because due to constant masticatory pressure vesicles and bullae get ruptured and it becomes ulcers and erosions. 1The diagnosis can be made histopathologically, clinically, and immunological methods.And thorough clinical history should be asked from the patient which includes presence of vesicles and bullous anywhere else in the body like skin, genitalia, and eyes.Since many oral lesions can cause lesions in the dermatological regions.Vesicle is defined as a superficial blister, 5 mm or less in diameter usually filled with clear fluid.And bulla is defined as a circumscribed collection of free fluid greater than 5 mm.In this article various procedures have been explained to diagnose the condition of vesiculo bullous lesions.He related that after rubbing the skin of the patient who was affected by pemphigus, there was a blistering or denudation of the epidermis with a glistening moist surface underneath. 3this was later confirmed by Lyell who described Nikolsky's sign in patients with toxic epidermal necrolysis.It is classically seen in pemphigus vulgaris.However other lesions showing sign for this are pemphigus foliaceus, graft versus host disease, paraneoplastic pemphigus, epidermolysis bullosa, oral lichen planus, bullous pemphigoid, mucous membrane pemphigoid, chronic erythema multiforme, dermatomyositis . 4this test cannot e performed in oral cavity because the blisters get ruptured.In oral cavity, after applying mucosal pressure, when ulceration or blisters appears, the test is said to be positive.Mucosal pressure can be by blowing air or using a blunt instrument or by finger.

METHOD
Done by applying lateral pressure with the index finger which gives shearing force to disrupt the intercellular adhesion 5 Biopsy- 6,7 Factors to be considered 1.
Ulcerated tissue site to be avoided ( because roof will not be present and sometimes the site may be masked by secondary inflammation and necrosis) 2.
To stop topical steroid ( in order to prevent false negative results) 3.
Two biopsy specimens from the affected site ( one specimen to be kept in 10% neutral buffered formalin for H & E staining and other is submitted in michel's medium for DIF) 4.
Sample of the patients serum or blood 5.
Sample/ lesions collected to be fresh (less than 24 to 48 hrs old) Tzanck test 8,9 Tzanck in 1947, used cytology for diagnosis of VB disorders particularly herpes simplex.It is a very simple and rapid technique Samples taken should be fresh Procedure for Tzanck test 1.
Cleaned and dried area 2.
At the base of the blister a sterile needle is inserted 3.
Then smear is taken from the blister which contains acantholytic cells 4.
Smear is then prepared on a clean glass slide and stained using Leishman stain 5.
The smear shows the presence of Tzanck cells which are formed during detachment Tzanck cell is a large round keratinocyte with a hyperchromatic nucleus and peripheral condensation of the chromatin Indication • giant cells identification that accompany in viral infections • Acantholysis, in case of pemphigus.

Le test or le cell inclusion phenomenon
Hargraves was the one who first explained this for SLE.typical LE cell will develop if the serum from the patient suffering from SLE is added to buffy coat of the normal blood.LE cell may be neutrophils or macrophages that has engulfed the denatured nucleus of an injured cell and contains LE body.

Immuno fluorescence
IF is an antigen antibody reaction where the antibodies are labeled with the dye (fluorescent dye) and then the antigen antibody complex can be seen using UV microscope . 10coons developed IF.Used for detection of antigens in the tisues.Gold standard technique for detection of autoimmune blistering diseases is through DIF.
Indirect immunofluorescence

Principle of fluorescence
An atom or molecule absorbs a quantum of light, an electron jumps to a higher energy level, thus displaces an electron from its shelf.When this displaced electron returns backs to its original ground state, it emits a quantum of light.This phenomenon is called photoluminescence and is of two types: Fluorescence and phosphorescence.Fluorescence is the property by which when illuminated by a light certain substances of certain wavelength, reemit the light to a longer wavelength.In phosphorescence, even after the exciting light is cut off, emission continues to persist.

Immunofluorescent Techniques Direct Immunofluorescence
It is the one step procedure in which fluoresceinated antibodies are applied to the frozen section of the skin.
View with UV microscope Indirect immunofluorescence 1.
Apply patients serum, antibodies bind to homologous structures in the section of monkey oesophagus 2.
View with UV microscope

Salt split technique
The purpose of this technique is to differentiate between two similar immunologically mediated disease having similar clinical features (case of bullous pemphigoid and epidermolysis bullosa) 11 It is of two types-direct and indirect 12 Direct technique is performed on freshly taken patient skin biopsy or by routine DIF, the one that has previously been investigated, whereas in indirect technique, substrate used is a normal human skin, cryocut sections are prepared after artificially inducing the junctional split, and then IDIF with patient's serum is carried out.

Elisa and western blot technique
For diagnosis of pemphigus vulgaris and foliaceus.This technique can detect antibodies to desmoglein 1 and 3 1.
To the surface of plastic tubes, absorb antigen and then excess Ag is removed by washing 2.
Antiserum of the patient is added and excess Antibody is removed 3.
Enzyme linked alkaline phosphatase is added and excess removed and incubated at 37 c 4.
Finally corresponding substrate p-nitro phenyl phosphate is added 5.
Optical density of yellow product is measured by spectrophotometer

CONCLUSION
Diagnosing autoimmune VB disease still remains in a dilemma.Immunoprecipitation, Western blot analysis, and elisa are the newer techniques which have evolved and gradually being used in the domain of immunobullous diseases.However, these investigations are complex, expensive, and more time consuming.The gold standard in diagnosing VB lesions is IF as it simple, reproducible, and less time is consuming technique.