RU2476879C1 - Differential diagnostic technique for lymphoproliferative dermatopathies by morphometric profile of basal keratinocytes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: histological examination of injured skin biopsy materials is performed by optical microscopy, immunophenotyping of infiltrate cells. If lymphomas and pseudolymphomas show a similar pattern described as manifested acanthosis, hyperkeratosis, along with dermal vascular distention, perivascular lymphohistiocytic infiltrates with lymphocytes penetrating into epidermis, as well as the same immunophenotype of the CD3⁺, CD4⁺, CD43⁺, CD45RO⁺ infiltrate cells; it is added with morphometry of basal keratinocytes in the histological preparations of the injured skin biopsy materials. It is followed by measuring their area and nucleocytoplasmic relation, and if observing an increase of the basal keratinocyte area in 1.5 times and more, and of the nucleocytoplasmic relation of basal keratinocytes in 2 times and more as compared to normal skin, skin lymphomas are diagnosed, while the unchanged morphometry of basal keratinocytes - skin pseudolymphomas.

EFFECT: use of the invention provides more accurate differential diagnosis of lymphomas and pseudolymphomas.

6 dwg, 1 tbl, 2 ex

Description

The invention relates to medicine, namely to dermatology, and can be used for the histological method of differential diagnosis of lymphoproliferative skin diseases, which belong to the heterogeneous group of dermatoses, due to the possibility of the development in the skin of a variety of benign lymphoid hyperplasias (pseudolymph) simulating malignant neoplastic processes (lymphomas )

A known method for diagnosing T-cell lymphomas of the skin is to conduct a cytological analysis of a blood smear taken from a patient, which is fixed, ribonuclease treated, stained with gallocyanin-chromium alum, microdensitometric lymphocyte interphase chromatin based on the obtained parameters (OD4 is the optical density of euchromatin; FORM_P is the nucleus form factor; IOD2 is the integrated optical density of heterochromatin; AREA is the area of the nucleus), the indicator is calculated: DF = -0.14379 * OD4 + 0.00003 * IOD2 + 10.4952 * FORM_P + 0.0001 * AREA-11, 1241 and with negative s the onset of this indicator is diagnosed with T-cell lymphoma of the skin [patent RU 2104526, 1998]. This method allows to detect malignant forms of lymphocytes in the blood, establish a diagnosis before the development of pronounced clinical manifestations, allows differential diagnosis of erythrodermic conditions in T-cell lymphomas of the skin and benign inflammatory dermatoses, but does not take into account morphological changes in the skin with these diseases, does not allow differentiate skin lymphomas from pseudolymphomas.

A known method of clinical and laboratory diagnosis of lymphoproliferative skin diseases, based on the immunophenotyping of infiltrate cells [Clinical and laboratory diagnosis of skin lymphomas. Medical technology No. FS-2006/299-U of October 26, 2006. Federal State Institution “Ural Scientific Research Institute of Dermatovenerology and Immunology”, Yekaterinburg // “Bulletin of Dermatology and Venereology”. - 2009. No. 4. S.27-34]. The authors suggest at the first stage of the examination to conduct a study of the anamnesis, features of the process, clinical manifestations, clinical monitoring, general clinical research methods. The result of the first stage of the examination is a preliminary diagnosis of benign dermatosis, primarily malignant lymphoma of the skin, lymphoproliferative skin diseases. At the second stage, the authors conduct a pathomorphological study of the biopsy of the skin, lymph node, bone marrow in a light-optical microscope, immunophenotyping of infiltrate cells using monoclonal antibodies (MCA): CD 45 (LCA), CD45RO, CD45RA, CD3, CD8, CD20, CD30 (ki1), ki 67 (company Becton Dickinson), after which the final nosological diagnosis of malignant lymphoma of the skin is established. However, given the high cost characteristics of the method, the need to use special equipment, the method is not always acceptable in practical health care and can only be carried out in specialized regional scientific centers of the 5th qualification level. In addition, the cells of lymphoid infiltrate undergo differentiation in the epidermis, changing their phenotype [Burg G., Zwingers T., Staegemeiz E. et al. Intrarater variabilities in the evolution of cutaneous lymphoma project group // Derm. Clin. - 1994. - Vol.12 - p. 311-314]. Burg G. showed that in the affected skin of patients with pseudolymphomas, with benign chronic dermatoses, such as small and large plaque parapsoriasis, clonal lymphocyte remodeling with a change in phenotype is possible [Burg G. Atlas of cancer of the skin. - New York. - 2006. - 368 p.].

In the described methods, immunophenotyping of the cells of the infiltrate makes it possible to assess the nature of T and B cell lines, makes it possible to diagnose skin lymphomas in the late stages of the disease, but does not solve the problems of early differential diagnosis of the initial manifestations of lymphomas from pseudolymph of the skin and inflammatory dermatoses. Our counterarguments are consistent with the data of other authors [Molochkov V.A., Lezvinskaya E.M., Molochkov A.V. Pseudolymphomas-prelimphomas-skin lymphomas // Russian Journal of Skin and Sexually Transmitted Diseases. - 2004. No. 5. C.4-7].

The prototype of the invention is a method for histological diagnosis of skin lymphomas by microscopic examination of biopsy samples of affected skin [Rodionov AN, Barbinov VV, Kazakov DV Histological diagnosis of skin lymphomas. - St. Petersburg. - 1997.283 s.]. The authors conduct histological diagnosis of skin lymphomas at the light-optical level, based on the following basic morphological criteria: 1) a change in the structure of the epidermis - the presence of acanthosis with wide, sometimes merging epidermal processes; vacuole dystrophy of basal keratinocytes; an increase in the figures of pathological mitoses in the epidermis; 2) the presence in the dermis of polymorphic cell proliferate, including atypical lymphocytes; the presence of diffuse or focal (Potria abscesses) penetration of lymphocytes into the epidermis.

However, the histological picture can be very variable depending on the clinical manifestations and stage of the disease, while the diagnostic value of the above criteria is not the same, it is not always reliable [Molochkov V.A., Kovrigina A.M., Ovsyannikova G.V. T-cell lymphomas of the skin: modern approaches to clinical and morphological diagnostics according to the classification of WHO / EORTC 2006 // Russian journal of leather-veins. diseases. - 2009. No. 2. S.4-15]. A number of pathological changes, such as the intensity of inflammatory reactions, atrophy and hypertrophy, reactive states, dysplasia and the definition of borderline conditions, the degree of decrease in tumor differentiation, and many other problems of detailing the histological diagnosis are solved differently taking into account the experience, knowledge, and qualification of a specialist on the one hand , inexhaustible variety of tumor structures and certain boundaries of the morphological method with another [Skrek S.V., Rodionov A.N., Raznatovsky K.I. Modern classification of skin lymphomas. Characteristics of the immune status of lymphoid cells in patients with various types of skin lymphomas // Clinical Dermatology and Venereology. - 2008. No. 2. p. 82-88]. The prototype of the invention takes into account the morphological changes that occur in the skin with lymphoproliferative skin diseases visually, but does not include immunophenotyping of the cells of the infiltrate, does not allow to objectify and differentiate the changes observed in the skin with lymphoproliferative skin diseases, and does not allow differential morphological diagnosis of lymphomas from pseudolymphs.

The objective of the invention is the objectification and differentiation of changes observed in the skin during lymphoproliferative skin diseases, differential morphological diagnosis of lymphomas from pseudolymph of the skin to determine the tactics of adequate treatment.

EFFECT: increased accuracy of diagnosis of lymphoproliferative skin diseases due to differential diagnosis of lymphomas and pseudolymph of the skin.

The proposed method for the differential diagnosis of lymphoproliferative skin diseases is as follows. A histological examination of biopsy samples of the affected skin, immunophenotyping of the cells of the infiltrate is carried out [Tsvetkova G.M., Mordovtsev V.V. Pathomorphological diagnosis of skin diseases. - Moscow, Publishing House of Medicine. - 1996. - 263 p.]; Kungurov N.V., Kokhan M.M., Sazonov S.V. et al. Clinical and laboratory diagnostic algorithms for malignant skin lymphomas // Clinical Dermatology and Venereology. - 2002. No. 6. p.16-19]. For light microscopy, skin biopsy samples are fixed in a 10% solution of neutral formalin (pH-7.3). After dehydration in ascending alcohols, it is poured into paraffin blocks, sections 4 μm thick obtained from the blocks are stained with hematoxylin and eosin, viewed under a light microscope, and morphological changes are described. For immunohistochemical studies, repeated sections from paraffin blocks are pre-treated in a water bath [Izban KF, Hsi ED, Ackan S. Immunohistochemical analysis of mycosis fungoides on paraffin - embedded tissue sections. // Mod. Pathol. - 2008. - V.11. p.978-982]. We used a Novocastra CD1a antibody panel, a monoclonal antibody panel CD2 ⁺ , CD3 ⁺ , CD4 ⁺ , CD5 ⁺ , CD45RO ⁺ , CD68 ⁺ , p69 ^{+ from} the Fast immune kit from Becton Dickinson, USA. The results of immunohistochemical reactions are visualized using the Enviston (Dako) system. The results are interpreted according to the WHO / EORTC classification [Molochkov V.A., Kovrigina AM, Ovsyannikova G.V. Skin T-cell lymphomas: modern approaches to clinical and morphological diagnostics (according to the WHO / EORTC classification) and treatment // Ross. journal skin veins diseases. - 2009. - No. 3. - C.4-9; Belousova, I.E., Kazakov D.V., Krivolapov Yu.A. Modern approaches to the diagnosis and treatment of primary skin lymphomas based on the new WHO-EORTC classification of B-cell skin lymphomas. // Archive of pathology. - 2007. - No. 6 - S.48-50]. Microphotography is carried out using a Leica DFS 420 camera and processed using a Leica Qwin standart v3.1.0 computer image analysis package. Statistical data processing is carried out using Statistica 7.0 using non-parametric statistics methods. In a similar histology described in lymphomas and pseudolymphoma how pronounced acanthosis, hyperkeratosis, a dermal vasodilation, perivascular lympho-histiocytic infiltrations penetration lymphocytes in the epidermis, the same immunophenotype infiltrate CD3 ^{+ cells, CD4 +, CD43 +, CD45RO} + lymphomas and pseudolymphomas, additionally carry out a morphometric study of basal keratinocytes in histological preparations of biopsy samples of the affected skin using a computer combined with a light microscope "Leica" company "L eitz Biomed ”(Germany) using an eyepiece micrometer and an ocular reticle (at 100 intersection points) [G. Avtandilov Computer morphometry. - Moscow. Publishing House of Medicine. - 1999.320 s.].

The area and nuclear-cytoplasmic ratio of basal keratinocytes are measured, and with an increase in the area of basal keratinocytes by 1.5 or more times compared to normal skin, the nuclear-cytoplasmic ratio of basal keratinocytes by 2 or more times compared with normal skin is diagnosed with skin lymphomas, and with unchanged morphometric indicators of basal keratinocytes - pseudolymphomas of the skin (table).

The invention is illustrated by the following drawings: figure 1 presents a photo of patient D. according to example 1; figure 2 - histogram of patient D. according to example No. 1; figure 3 is a fragment of a histogram of patient D. according to example 1; figure 4 - photo of patient U. according to example No. 2; figure 5 is a histogram of patient U. according to example No. 2; figure 6 is a fragment of a histogram of patient U. according to example No. 2.

The authors in the available scientific and medical and patent literature did not find information on the use of morphometric studies with the measurement of the area and nuclear-cytoplasmic ratio of basal keratinocytes for the differential diagnosis of lymphomas from pseudolymph of the skin. Thus, the claimed invention meets the patentability criterion of "novelty."

The authors' research was the first to prove the possibility of differential diagnosis of lymphomas from pseudolymphs of the skin by morphometric studies with the measurement of the area and nuclear-cytoplasmic ratio of basal keratinocytes. Thus, the claimed invention meets the criterion of "inventive step".

The proposed method of differential diagnosis is illustrated by the following examples.

Example No. 1. Patient D., 56 years old, an employee of a chemical oil refining complex, was admitted to the clinic of the Republican Dermatovenerologic Dispensary (RCVD) for inpatient treatment with complaints of rashes throughout the skin, intolerable itching. She considered herself a patient for a year, repeatedly contacted the police department at the place of residence, where desensitizing, symptomatic therapy was prescribed, which did not lead to clinical improvement. To clarify the diagnosis, she was sent to the RKVD with guiding diagnoses: skin lymphoma? pseudolymphoma of the skin? Upon admission, the skin-pathological process was widespread, in the form of spots of pink-red color, of various sizes and shapes, with a rougher than flaky surface (Fig. 1). Peripheral lymph nodes were enlarged to the size of beans. Histologically, in the biopsy specimens of the affected skin, pronounced acanthosis with wide, fused epidermal processes, hyperkeratosis, and horn invaginations was noted. In the dermis there were dilated vessels lymphohistocytic perivascular infiltrates with the penetration of lymphocytes into the epidermis (figure 2). An immunophenotypic study using a panel of monoclonal antibodies established the immunophenotype of infiltrate cells: CD3 ⁺ , CD4 ⁺ , CD43 ⁺ , CD45RO ⁺ , which did not allow to establish a final diagnosis. To clarify the diagnosis and differential diagnosis of lymphoma from pseudolymphoma of the skin, a morphometric study of basal keratinocytes was carried out (figure 3).

The area of basal keratinocytes in this patient was 60.15 μm ² , the nuclear cytoplasmic ratio was 0.49, which was 1.5 and 2.2 times higher than that in healthy skin (normal), respectively. Based on a comprehensive clinical and laboratory immunophenotypic, histological and morphometric study, this patient was diagnosed with skin lymphoma (fungal mycosis stage I). After receiving 2 courses of specific prospidinotherapy, the elements regressed. Relapse of the disease during the year was not observed.

Example No. 2. Patient U., 67 years old, a pensioner, was admitted to the RKVD clinic with presumptive diagnoses of skin lymphoma? pseudolymphoma of the skin? Upon receipt, she complained of severe constant itching, chills, fever up to 38.5 ° C, redness of the entire skin, its detachment. She considered herself sick for a month, when after treatment in the Yakty-Kul sanatorium, where she received mineral baths and mud therapy for sciatica, after 8 procedures, redness of the skin of the body appeared. Objectively upon admission: a universal process, the type of erythroderma (figure 4). The skin is bright red with a livid shade, infiltrated, lichenified in the area of the skin folds, abundantly covered with large plate scales. Peeling is especially pronounced on the lower extremities, where there were numerous excoriations and crusts. Hyperkeratosis was noted on the palms and soles. The submandibular, cervical, axillary, and inguinal lymph nodes were enlarged to 3 cm in diameter, with a dense-elastic consistency upon palpation, mobile, painless. Histological examination revealed: pronounced acanthosis, parakeratosis and hyperkeratosis in the epidermis. In the dermis, the vessels are dilated. Perivascular lymphocytic infiltration was observed with the penetration of lymphocytes into the epidermis (figure 5). Immunophenotype of CD3 ⁺ , CD4 ⁺ , CD43 ⁺ , CD45RO ⁺ infiltrate cells. Morphometric studies of basal keratinocytes (Fig.6) deviations of the parameters from the norm did not reveal: the area of the basal cells was 42.5; nuclear cytoplasmic ratio of 0.23. Given the anamnestic data, clinical and histological picture, morphometric studies, the diagnosis of pseudolymphoma syndrome was established. The patient underwent detoxification therapy, including a course of plasmapheresis in combination with enterosorbents and antihistamines. Hyperemia and infiltration became less pronounced, peeling was absent, areas of healthy skin appeared, lymph nodes decreased, the temperature returned to normal. It was possible to achieve clinical cure after a 2-fold injection of diprospan.

As can be seen from the above examples, the diagnosis of lymphoproliferative skin diseases is difficult, clinical and laboratory research methods are needed. Conducting histological studies immunophenotyping cells dermal infiltrate showed similar histological picture is described as pronounced acanthosis, hyperkeratosis, dilated blood vessels, perivascular lymphohistiocytic infiltration with infiltration of lymphocytes in the epidermis, and similar phenotype ^{CD3 +, CD4 +, CD43 +} , CD45RO +, as in lymphomas , and with pseudolymphomas of the skin. The use of morphometric studies of basal keratinocytes made it possible to differentiate skin lymphoma from pseudolymphoma, which allowed, in turn, to prescribe adequate treatment, which led to a regression of the disease. Therefore, the diagnosis

lymphoproliferative skin diseases should be carried out using the entire spectrum of diagnostic methods: clinical, histological, immunophenotypic and morphometric.

Thus, compared with the prototype, the proposed method for the differential diagnosis of lymphoproliferative skin diseases according to the morphometric profile of basal keratinocytes allows:

a) use the whole range of diagnostic methods of lymphoproliferative skin diseases that currently exist, including clinical and histological methods of investigation, immunophenotyping of infiltrate cells;

b) objectify and differentiate the changes observed in the skin during lymphoproliferative skin diseases by means of morphometric studies of basal keratinocytes;

c) carry out differential diagnosis of lymphomas from pseudolymphs of the skin by measuring the area and nuclear-cytoplasmic ratio of basal cells.

The proposed method was a differential diagnosis in 37 patients with skin lymphomas and 24 with pseudolymphomas, and in all cases the specified technical result was achieved. The method is reproducible in a histological laboratory of a hospital. Thus, the claimed invention meets the patentability criterion of "industrial applicability".

Table Morphometric profile of basal keratinocytes in lymphoproliferative skin diseases Nosology Area of basal keratinocytes (μm ² ) Nuclear cytoplasmic ratio in basal cells Skin lymphomas (n = 37) 64.2 ± 4.64 ** 0.47 ± 0.03 *** Pseudolymphomas of the skin (n = 24) 42.6 ± 4.74 0.24 ± 1.01 Control (healthy skin) (n = 18) 40.1 ± 2.26 0.22 ± 0.04 Note: ** - significance of differences in indicators with control at p <0.01; *** p <0.001, in some cases p> 0.05.

Claims (1)

A method for the differential diagnosis of skin lymphomas from pseudolymphs, including histological examination of biopsy samples of affected skin by light microscopy, characterized in that they additionally carry out immunophenotyping of the cells of the infiltrate, with a similar histological picture, including lymphomas and pseudolymphomas, pronounced acanthosis, hyperkeratosis, the presence of vascular expansion in the dermis, perima lymphohistiocytic infiltrates with the penetration of lymphocytes into the epidermis, as well as the same immunophenotype of infiltrate cells ata, ^{namely: CD3 +, CD4 +, CD43} +, CD45RO +, additionally performed morphometry basal keratinocytes, measure their area, and nuclear-cytoplasmic ratio and with an increase in basal keratinocytes area 1.5 times or more as compared with normal skin , the nuclear-cytoplasmic ratio of basal keratinocytes in 2 or more times compared with normal skin is diagnosed with skin lymphomas, and with unchanged morphometric parameters of basal keratinocytes - skin pseudolymphomas.

Images