Psoriasis is a polygenic, immune-mediated, chronic, recurrent inflammatory skin disease.Drip psoriasis is one of the psoriasis vulgaris, and its clinical manifestations are mainly erythema, papules and scales.The etiology of pityriasis roseum is not clear, and some scholars think it is related to the infection of human herpes virus type 6 (hhv-6) and human herpes virus type 7 (HHV7), which is also a erythema papular and squamous skin disease.Both psoriasis droplet and pityriasis rosacea occur in the trunk and have many similarities to the naked eye.Typical pitchoriasis and pityriasis rosae are easy to identify, the former has the characteristics of scraping wax phenomenon, membrane phenomenon and punctate bleeding, and the latter rash’s long axis is consistent with the direction of skin grain, and part of pityriasis rosae can be identified with the mother spot.However, it is difficult to distinguish the atypical droplet psoriasis and the atypical pityriasis rosae by naked eyes, so the pathological examination of skin tissue is a valuable diagnostic method.However, the pathological examination of skin tissue is a wound sampling method, and the patient compliance is poor.We identified these two diseases according to different cytological types of skin rash surface of psoriasis droplet and pityriasis rosacea, and used non-invasive sampling method to paste skin exfoliated cells in skin rash area with transparent tape, and the experimental results were satisfactory.
1.1 Research object
The study subjects were all patients who went to the dermatology department of our hospital from September 2015 to December 2016, and conducted histopathological examination on skin lesions of patients with atypical psoriasis and patients with pityriasis rosacea, and signed informed consent forms.Thirty patients with psoriasis punctiform and pityriasis rosaceae were selected, including 18 males and 12 females in the psoriasis group (age 39.50±21.55).There were 14 males and 16 females in the pityriasis group, aged 32.33±23.75.The gender composition ratio and age of the two groups were comparable.
1.2 Reagents and materials
HE dye solution, the primary antibody was mouse anti-human granulocyte myeloperoxidase antibody, the secondary antibody was horseradish peroxidase-labeled rabbit anti-rat IgG polyclonal antibody, and diaminobenzidine (DAB) was the substrate color developing kit, all purchased from Bio Genex company in the United States.EAF fixing solution configuration: take 5mL glacial acetic acid, 40% formaldehyde 10mL, anhydrous ethanol to 100mL.
1.3 Method
1.3.1 Specimen collection
The patients in both groups were pasted on the rash site with commercial transparent adhesive tape, and then removed and discarded after 3s (removing dry cells from the surface). Then three adhesive tapes were pasted on the same site for experimental use.The tapes with specimens were stained with HE (removal of the first 2 steps of xylene dewaxing) and stained with immunohistochemistry, and the tissue cytology was performed.At the same time of exfoliating cells sampling for each patient, non-transparent adhesive tapes of the two groups of patients were pasted on the rash area to select one site for skin histopathological examination, and HE staining was performed.
1.3.2 Specimen
With HE staining, adhesive tape with detached cell specimens was fixed on the slide, and the specimens and markers were made with the cell side up.One of the specimens was fixed with 95% alcohol, and modified HE staining method, namely, the staining method of cervical epidermal cells, was adopted. After dehydration and transparency, neutral gum was sealed and observed under light microscope.
1.3.3 Immunohistochemical staining
The remaining two specimens were fixed with EAF fixator at 4℃ for 15min, washed with PBS, and operated according to the kit instructions.One specimen was used as the blank control, and the other specimen was treated with DAB and HE restaining for 40s respectively.Dye and dehydrate, and seal with neutral resin for observation under light microscope.
1.3.4 Skin specimens
See histopathology operating procedure of HE staining.
1.4 Standard of diagnosis
In the HE staining results of the tape specimens, cell types were determined by the nuclear morphology under optical microscope, and the nuclei presented as lobes or rods were judged as neutrophils, while the round or nearly round nuclei were skin squamous cells, which was equivalent to keratinocytes or granular layer cells in the cuticle by the pathological HE staining method of skin tissue.Meanwhile, the distribution of neutrophils was observed.Microscopically, neutrophils with clustered distribution were positive and diagnosed as psoriasis.The absence of neutrophils or the presence of a small number of scattered neutrophils was negative.If neutrophils were found to be clustered in the exfoliated cells, the results were positive and the psoriasis was diagnosed as droplet psoriasis.If the diagnosis was not cluster distribution (scattered distribution of neutral cells + no neutral cells), the result was negative and the diagnosis was pityriasis rosea.The diagnosis of transparent adhesive tape method in the diagnosis of psoriasis (%) = a/(a + b), pityriasis rosea diagnostic rate (%) = d/(c + d), overall diagnostic accuracy (%) = (a + d)/(a + b + c + d).
1.5 Statistical treatment
SPSS13.0 software was used to compare the coincidence rate of the diagnosis results of scotch tape with the histopathological HE staining and the distribution of neutrophils in the two groups. The differences were statistically significant with P < 0.05.
2.1 Scotch tape
The distribution of neutrophils in skin lesions of the two groups was shown in table 1, and the comparison of diagnosis rate of shedding cells and histopathological diagnosis was shown in table 2. The incidence of neutrophils in the two groups was statistically significant (chi-square =52.50, P < 0.01).After HE staining, the distribution of detached cells under the microscope was shown in figure 1 ~ 3.
2.2 Immunohistochemical staining results
Neutrophils were further confirmed to be neutrophils because neutrophils contain myeloperoxidase.
2.3 Comparison of diagnostic coincidence rate
Are shown in table 2.The coincidence rate of the transparent tape cell method was 93.33% and 100.00%, respectively, and the total diagnostic accuracy was 96.67%.
Demodex scabies, sarcoptic mange and superficial fungi have been used for many years.However, it is rarely reported that skin diseases are diagnosed by pasting skin exfoliated cells with scotch tape.In recent years, Chinese scholars Chen xiangming et al. took the lead in the study of using transparent tape to paste epidermal exfoliated cells in the rash area to distinguish atypical patchy psoriasis from atypical numeral eczema, and obtained satisfactory results in a small sample.In this study, detached cells in rash area were used to identify atypical pityriasis and atypical pityriasis rosacea.We know that no matter in advanced psoriasis or static psoriasis, the most key and specific histopathological characteristics of psoriasis skin lesions of all types are neutrophils aggregation in the upper part of keratinosis, namely Muro microabscess, and Muro microabscess in drip psoriasis can be hump-like.The mechanism of Muro microabscess is closely related to the chemotaxis of neutrophils induced by il-8.The unique histopathological characteristics of Muro microabscess provide theoretical and experimental basis for the diagnosis of psoriasis by skin exfoliation cell method with adhesive tape.However, focal keratinosis was found in the epidermal layer of furyriasis rosea histopathologically, and there was usually no Munro microabscess formation, which was also confirmed in the transparent adhesive strip cell exfoliation method of patients diagnosed with furyriasis rosea histopathologically.
Histopathological diagnosis of psoriasis punctate and pityriasis rosae was the gold standard for the diagnosis of the two diseases (i.e., the diagnostic rate was 100.00%).The results showed that the diagnostic rate of psoriasis was 93.33%(28/30) and pityriasis rose was 100.00% (30/30), with a total accuracy of 96.67%.The results show that the method is very close to the gold standard diagnostic method, which indicates that the study has important clinical application value.
In this study, immunohistochemistry confirmed that all the rod nuclei and lobulated nuclei of these clusters were neutrophils, indicating that HE staining method can be used independently for the characterization of neutrophils, so that immunohistochemistry staining is not necessary in clinical practice, which can significantly reduce the testing cost.About 2 cases of neutrophils in patients with psoriasis guttata with a scattered distribution, it may be related with the paste adhesive tape from skin rashes, not stick to neutrophils in parts of the cluster area, or with a disposable materials just paste the parakeratosis layer, and the omission of the following is a microabscess Muro layer, so suggest paste 2 times in a row, the rash area may improve the positive rate.In addition to psoriasis, psoriasis somalis, pyoderma, pustules, candidiasis, seborrheic dermatitis, and syphilis, neutrophils infiltrate the epidermal stratum corneum.However, these diseases can be identified in clinical manifestations and other laboratory tests and are not within the scope of this study.
The skin exfoliating cell method of skin rash sticking with transparent tape is applicable to exclude other similar skin diseases only when the difference between atypical psoriasis and atypical pityriasis rosea is not possible.Although the exfoliated cells diagnostic method is not able to see the pathological conditions of epidermis, dermis and subcutaneous tissues longitudinally like the skin histopathology, it has the advantages of simple operation, low test cost and high patient compliance, so it has further research and application value.
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