Location And Significance Of Type IV Collagen In The Bullous Pemphigoid

Background:Bullous Pemphigoid(BP) is a kind of chronic and systemic subepidermal bullous dermatosis. Early skin lesions are mostly blisters whose rare is 70%; a few cases are blisters with erythemas and papules.Most of the cases are slow onset and the blisters are limited to certain part;half of the cases abruptly involve the whole body and a few cases involve the whole body at the beginning. Generalized Bullous Pemphigoid is a kind of very severe disease.BP'histopathology is subepidermal bullous. A lot of eosinophils mixed with mononuclear cells and neutrophils often infiltrate perivascular at the dermis in the uninflammatory bullous. There is epidermis intracellular edema and eosinophils extravasate to spongiosis in the epidermis in the inflammatory bullous.there is often eosinophile spongiosis nearby the bullous'erythema region . It is a little difficult to diagnose BP only using HE staining in the practical clinical observation .Because the subepidermal bullous of the HE staining are seen in many diseases in pathological tissues.We should use not only clinical manifestations and histology ,but also immunofluore- scence and immune peroxidase antigen-targeted method for the definite diagnosis of subepider- mal bullous. Immunofluorescence can locate the certain part of the dermal epidermal separation. However, needing special fluorescent microscope and fluorescence quenching easy and difficult long-term preservation of specimens and photographic record to be and the need for frozen section machine and so on,are the disadvantages of immunofluorescence . Therefore more difficult to carry out in the general laboratory. Abroad have reported that the use of paraffin-embedded specimens of immune peroxidase antigen-targeted,which method is the use of lesions of paraffin-embedded biopsy specimens using immunohistochemical peroxidase antigen directly targeted technology to determine the dermal epidermal separation of the specific site,is an alternative of salt split skin immunofluorescence examination. This method can be positioning part of a number of known basement membrane components such as keratin 5 / 14, laminin and typeⅣcollagen at the top or bottom of the blister, which will be the location of blister classified as basal cells, lamina lucida, or under lamina densa. Such as , all the depositions of immune response are at the bottom of the blister in epidermolysis bullosa simplex. Keratin positioning at the top of blisters, and laminin and typeⅣcollagen positioning at the bottom of blister in BP. In dystrophic epidermolysis bullosa, acquired epidermolysis bullosa and bullous systemic lupus erythematosus, the immune response of these three materials are deposited on top of blisters. At present, not yet see a similar immunohistoche- mical method of diagnosis of BP reported in the literature.TypeⅣcollagen is one of the major components of basement membrane,whose antibody is used to identify the lamina densa (LD). This identification method is meaningful to distinguish the blister formation position in subepidermal vesicular diseases.Objective:To detect the location and significance of the location of type IV collagen in bullous pemphigoid.Materials and MethodsMaterials:Select samples: paraffin-embedded tissue samples are from the second hospital of Jilin University dermatology clinic for the last ten years under the diagnosis of pathological HE subepidermal blisters. A total of 63 cases, 42 cases of male, female 21 cases, and the youngest 15-year-old, maximum 87-year-old.Group 1: clinical and HE pathological Supporting BP diagnosis are 26 cases.Group 2: dermatitis, eczema, erythema multiforme and so on,witch are 13 cases easy to be confused in HE staining with BP, including clinical and pathological diagnosis of HE for the tropic of acute febrile disease that is sweet skin disease example 1 and erythema multiforme example 4, 1 cases of porphyria, lichen planus-like keratosis axample 1, two cases of atopic dermatitis, two cases of eczema, the other two cases of dermatitis.Group 3: 20 cases of HE pathology question except BP.Group 4: HE pathology and clinical both support epidermolysis bullosa Example 4.The main reagents: anti-1 (mouse anti-human collagen type IV monoclonal antibody); PV-9000 (two-step immunohistochemical detection reagent): 1 reagent (polymer-assisted agent, Polymer Helper-use), reagent 2 ( goat anti-rabbit / mouse IgG polymer, Polyperoxidase-anti- mouse / rabbit IgG-use); DAB color kit: Reagent A (condensed buffer), reagent B (enrichment DAB solution), reagent C (enrichment hydrogen peroxide solution); 3% H2O2 deionized water; pepsin digestive juice (that is, by type); PBS buffer.Methods:(1)Deal with the slides: The slides are immersed in 10% sulfuric acid overnight. Adequately washed in running water. Deionized water to fully wash. 100% ethanol overnight soaking. 37℃dry oven. Followed by Poly-L-Lysine coated after removed. Natural drying at room temperature, cartoning, spare.(2)Specimens of the general treatment: The paraffin-embedded tissue samples for thin-cut (4 microns), placed on the prior slide good deal, 37℃overnight dry, the specimens placed in xyleneⅠ1 hour, xyleneⅡdewaxing a half-hour, 95% ethanol, 100% ethanolⅠ,Ⅱall 15 minutes, five minutes of immersion in distilled water, PBS wash buffer 5 minutes×2 times.(3)Immunostaining steps①3% H2O2 deionized water were incubated for 10 minutes, PBS washed 2 minutes×3 times.②Adding dropwise stomach enzymes digestive juice to the tissue section (the stomach enzymes should be taken back into the refrigerator at once), 37℃water bath incubation for 5-10 minutes, PBS washes 3 times.③Adding dropwise anti-1 (mouse anti-human collagen type IV monoclonal antibody), 37℃water bath incubation for 1-2 hours, PBS washes 2 minutes×3 times.④Adding dropwise reagent 1 (polymer-assisted agent, Polymer Helper-use), 37℃water bath incubation for 20 minutes, PBS washes 2 minutes×3 times.⑤Adding dropwise reagent 2 (goat anti-rabbit / mouse IgG polymer, Polyperoxidase- anti-mouse/rabbit IgG-use), 37℃water bath incubation for 30 minutes, PBS washes 2 minutes×3 times.⑥DAB color about 10 minutes, observe the color situation under the microscope.⑦Fully washed in running water, hematoxylin- counterstaining, blue, and dehydrated, transparent, and mount.Results:(1)Group 1 (clinical and HE pathological Supporting BP diagnosis) has 26 cases in wicth 21 cases of immune peroxidase antigen-targeted showed that typeⅣcollagen located linear in the bottom of blisters, the positive rate was 80.8%, see Figure 5. 3-6. Group 2 (dermatitis, eczema, erythema multiforme and so on,witch are easy to be confused in HE staining with BP) 13 cases, the positive rate was 7.7%, see Figure 5.7-9. The positive rate of typeⅣcollagen of group 1 patients is significantly higher than group 2.It is statistical significance (P <0.01).(2)Group 3(HE pathology question except BP) Example 20, 11 positive cases(see Figure 5. 10-11), nine cases of negative. Positive results can be diagnosed as BP. The results of negative can be combined with clinical and the prognosis for further analysis and diagnosis; three cases with positive results in four cases which were originally diagnosed as dermatitis herpetiformis under HE pathological,these three cases should be corrected for BP.(3)Group 4: Epidermolysis bullosa Example 4, 2 cases of positive(see Figure 5. 12), 2 cases of negative. The two cases were positive at the bottom of the blisters, so they are considered to be epidermolysis bullosa simplex and junctional. The two negative cases are considered to be dystrophic epidermolysis bullosa.Conclusion:(1)According to the typeⅣcollagen linear distribution at the bottom of the blisters,the immunohistochemical method of using typeⅣcollagen for staining basement membrane can clearly diagnose bullous pemphigoid, which is easy to get rid of the possibility of confusion of the inflammatory and other allergic diseases,et al. This experimental method is simple, easy, low-cost and higher sensitivity and specificity in the diagnosis of bullous pemphigoid ,which can be an alternative of immunofluorescence. In addition, samples can be used in paraffin-embedded tissues and carried out retrospective study.This method is worthy of promotion to the clinical.(2)In clinical, the diagnosis of bullous pemphigoid should be combined clinical with histopathological and immunofluorescence or immunoperoxidase antigen-targeted to analysis and draw the right conclusions.