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DERMATOVENEREOLOGY
INVENTION
Patent of the Russian Federation RU2104526

METHOD OF DIAGNOSTICS OF T-CELLS BY LYMPHMOUS SKIN

METHOD OF DIAGNOSTICS OF T-CELLS BY LYMPHMOUS SKIN

The name of the inventor: Kogan Emmanuil Markovich ; Zhukotsky Alexander Vasilyevich; Kopylov Viktor Fedorovich; Leszvinskaya Elena Mikhailovna; Chernysh Sergey Anatolievich
The name of the patent holder: Kogan Emmanuil Markovich ; Zhukotsky Alexander Vasilyevich; Kopylov Viktor Fedorovich; Leszvinskaya Elena Mikhailovna; Chernysh Sergey Anatolievich
Address for correspondence:
Date of commencement of the patent: 1995.05.05

Use: The invention relates to medicine, namely, to dermatology and hematology, is intended for early diagnosis and differential diagnosis of T-cell lymphomas of the skin. A method for diagnosing T-cell skin lymphomas (erythrodermic variants) by cytological analysis of a biological material taken from a patient, characterized in that a blood smear is taken as a biological material, fixed, ribonuclease treated, stained with gallocyanin-chromic alum, microdensitometric interfacial chromatin of lymphocytes, Based on the parameters obtained (OD4 - optical density of euchromatin, FORM_P - core form factor, IOD2 - integral optical density of heterochromatin, AREA - core area), calculate the index: DF = -0.14379 P OD4 + 0.00003 IOD2 + 10.4952 Ч FORM_P + 0,0001 Ч AREA - 11,1241 and at negative value of this parameter diagnose T-cell lymphoma of the skin.

DESCRIPTION OF THE INVENTION

The invention relates to medicine, namely, to dermatology and hematology, is intended for early diagnosis and differential diagnosis of T-cell lymphomas of the skin.

Most often it is necessary to differentiate erythrodermic variants of T-cell lymphomas of the skin and erythroderma that occur in benign inflammatory dermatoses (eczema, psoriasis, neurodermatitis, etc.). In addition, up to the present time, there has been no assessment of changes in lymphocytes in patients with erythrodermal conditions, which are defined as pre-lymphocytes: exfoliative Wilson-Broca dermatitis and red Otryabridae of Gebra. In clinical practice, it is often necessary to address the issue of malignant transformation into lymphoma of chronic inflammatory dermatoses.

The existing methods of diagnosing T-cell lymphomas of the skin can not fully satisfy clinicians, since they allow us to establish a diagnosis only if there is a pronounced clinical picture that is laborious in execution (immunological, cytochemical, cytogenetic, etc.) and does not reliably reflect changes in the lymphocyte population characterizing malignancy Process.

One of the most well-known diagnostic methods is the detection of atypical forms of lymphocytes in patients with erythrodermia in malignant T-cell lymphomas. These erythroderms include the erythrodermic form of fungal mycosis and Cesari syndrome, which is currently considered as a leukemic variant of the erythrodermic form of fungal mycosis.

Previously, attempts were made to describe atypical T-lymphocytes (Cesari cells) in patients with erythroderma. However, each of the methods listed below has its drawbacks.

The histological picture of erythrodermic conditions does not always reflect the nosological specificity of the process, in view of the pronounced inflammatory component of the biopsy material. Often, for the diagnosis is required several biopsies, which is traumatic for the patient.

Light microscopy allows detecting atypical lymphocytes in peripheral blood only in advanced stages of the disease, in addition, since the analysis is performed visually, the result is a qualitative description of the morphology, which does not allow to objectify and quantify the results obtained [1].

Electron microscopic studies allow us to obtain general ideas about the distribution of chromatin in lymphocytes, but the technique does not allow us to evaluate the functional state of the genome, it is laborious enough, especially in preparing the material for the study [2],

Scanning electron microscopy can not be used in the diagnosis of erythroderma, as it does not allow to obtain information on changes in the nuclei of lymphocytes specific for diagnosis [3].

Cytochemical studies do not provide a clear difference in the content of enzymes in reactive T lymphocytes in benign inflammatory dermatoses and malignant T lymphomas, i.e. There are no specific cytochemical markers of Sesari cells [4].

Lymphocyte cytophotometry reflects their proliferative activity based on the definition of cells that include thymidine only in the S-phase and does not reflect the qualitative redistribution of chromatin in each cell [5].

Cytogenetic studies do not have strict specificity and do not always reveal pathological changes even with far-reaching processes [6].

Morphometry of lymphocytes shows that the degree of rupture of the nucleus, determined by the ratio of the circumference of the nucleus, is the index of the nuclear contour, which was the largest in Cesari cells and the smallest in lymphocytes in patients with chronic lymphocytic leukemia. But the application of only the method of classical morphometry does not allow to assess the fine structure of supramolecular organization of chromatin in the nucleus [7].

Immuno-phenotypic characteristics with the use of monoclonal antibodies that detect specific tumor markers on lymphocyte membranes are the most informative for diagnosing manifestations of T-cell lymphomas and differential diagnosis of them with benign inflammatory skin diseases. However, this technique requires the availability of expensive reagents, allows to diagnose T-cell lymphomas at later stages of the disease, when skin and lymphadenopathy are expressed than the method proposed in this application, and there are difficulties in preparing the material for the study, because Blood is taken from the vein, which is more traumatic for a patient with skin lesion [8].

This method can be considered as a prototype, because it is the most informative for diagnosis of T-cell lymphomas and differential diagnosis of them with benign inflammatory skin diseases.

The reaction is carried out in 3 stages.

I. Isolation and preparation of lymphocytes for reaction.

Blood sampling from the ulnar vein and lymphocyte secretion by the usual method on a density gradient of feco-verografen by centrifugation. The cells are counted in the Goriaev chamber.

II. Preparation and conduct of the indirect immunofluorescence reaction.

On clean defatted glass, apply the holes from the parafilm, 4 for each patient. Poly-L-lysine is applied to each well to fix the cells. The preparation is incubated in a thermostat for 40 minutes at 37 ° C. The reaction consists of successive incubations. First - with a suspension of cells, then the preparations are washed and monoclonal antibodies are applied to the wells. After washing to remove the antibodies, the preparations are incubated with an anti-mouse IgG antiserum, a fluorescein-treated isothiocyanate. Then, after washing, the preparations are poured with glycerin mixed with physiological saline (1: 1) and covered with coverslips.

III. Counting of subpopulations of T-lymphocytes in a luminescent microscope equipped with a phase-contrast device.

The ratio of cells having ring-shaped luminescence to the total number of cells counted in a phase-contrast device is expressed as a percentage, which reflects the content of certain phenotypic populations and allows the material to be assigned to be assigned to a specific nosological form (T-lymphoproliferative process). Diffusely-colored (dead) cells are not taken into account.

It is an object of the present invention to provide a method for earlier diagnosis and differential diagnosis of skin T-cell lymphomas (erythrodermic variants) and to reduce the traumatism of a patient.

The method is carried out as follows

Prepare a smear of peripheral blood taken from the finger of the hand, fix (for example, a mixture of Nikiforov: ethanol and ether in a ratio of 1: 1) for 15-20 minutes, air dried. Ribonuclease treatment is performed, ribonuclease (Reanal, BHP) is diluted in an osmolar solution of sucrose at a concentration of 200 IU / 100 ml. Treatment lasts 50 - 60 minutes at a temperature of 37 ° C, after which the smear is washed in 3 shifts of distilled water. The color is produced in a solution of gallocyanin-chromic alum (GCC), prepared according to the standard procedure [9] for 3-4 hours at 37 ° C. The preparation is washed for 4 to 5 minutes with standing tap water.

After staining, the preparation is covered with a cover slip and microdensitometric examination is carried out on the image analysis system. The result of microdensitometry is the optical, topological and geometric characteristics of interphase chromatin in components (euchromatin and heterochromatin) and the nucleus as a whole.

Previously, the most informative parameters of the structure of interphase chromatin were established on training samples:

OD4 is the optical density of euchromatin;

FORM_P - kernel form factor;

IOD2 - integral optical density of heterochromatin;

AREA is the area of ​​the core.

For a comprehensive evaluation of these indicators, the parameter is calculated: DF = -0.14379 Ч OD4 + 0.00003 Ч IOD2 + 10.4952 Ч FORM_P + 0.0001 Ч АREA - 11.1241.

The formula for calculating the DF parameter was obtained by applying a linear discriminant analysis [10] using a standard package of statistical programs. If the DF value is negative, T-cell lymphoma of the skin is diagnosed.

A similar connection between the numerical values ​​was established by examining the groups of patients treated in the MONICA skin clinic for T-cell lymphoma of the skin (11 people) and benign inflammatory dermatoses (psoriasis and neurodermatitis - 10 people). As a result of the analysis, the most significant parameters of the structure of interphase chromatin of peripheral blood lymphocytes were revealed, the derived parameter DF was calculated to detect T-cell lymphoma of the skin.

Example 1 . Patient B, 63 years old, N case history - 7646, MONIKI.

Entered the clinic with complaints of redness of the skin, itching, burning, chills.

Clinical diagnosis: T-cell lymphoma of the skin, erythrodermal stage.

During the treatment in the clinic, the chromatin of interphase nuclei of peripheral blood lymphocytes of the patient was investigated using the method proposed in this application, using the standard method for preparing a whole blood smear taken from the finger. The resulting smear was dried in air, fixed with a mixture of Nikiforov. Further, to eliminate the effect of ribonucleic acid on the scan results, ribonuclease treatment was performed. Ribonuclease was diluted in an osmolar solution of sucrose at a concentration of 2000 E per 100 ml of the solution. The preparations were treated in this solution for 1 hour at 37 ° C., after which they were washed in 3 changes of distilled water. Then, the preparations were stained in a solution of MHC. A solution of MCC is prepared according to a standard procedure [9]. Staining is carried out for 3 hours in a thermostat at 37 ° C, after which the preparations are washed 5 minutes with tap water.

The colored preparations were encased in Canadian balsam and, after drying, were examined on an image analysis system.

Parameters of the chromatin structure for the examined patient:

OD4 3,02,207; FORM_P - 0.82145; IOD2 - 39080.8; AREA - 7938.7.

Further, for a complex evaluation, the DF value of -0.9710674 was calculated,

The value of the indicator DF <0; Consequently, the patient has changes in the structure of interphase chromatin corresponding to T-cell lymphoma of the skin, which completely coincides with the data of clinical and laboratory studies.

Example 2 . Patient V., 40 years old, N case history 14762, MONIKI.

Entered the MONICA skin clinic with complaints of reddening of the skin, more on the lower extremities, itching.

Clinical diagnosis: Neurodermatitis.

Using the proposed method, the structure of the chromatin of peripheral blood lymphocytes of the patient is examined (see Example 1.).

Parameters of the chromatin structure for the examined patient:

OD4 - 4,48286; FORM_P - 0.938331; IOD2 - 74411.4; AREA - 13686.

Further, for the complex estimation, the indicator DF = 1.6802235 was calculated.

The value of DF> 0, therefore, the revealed changes in the structure of chromatin correspond to changes in benign inflammatory dermatoses.

Example 3 . Patient G., 56 years old, N case history - 1497; MONIKI.

Entered the clinic with complaints of redness of the skin, chills.

Clinical diagnosis: psoriasis.

Using the proposed method, the structure of the chromatin of peripheral blood lymphocytes of the patient is examined (see Example 1.).

Parameters of the chromatin structure for the examined patient:

OD4 - 3.26156; FORM_P - 0.933456; IOD2 79238.4; AREA - 14092.6.

Further, for the complex estimation, the indicator DF = 1.9901397 was calculated.

The value of DF> 0, therefore, the revealed changes in the structure of chromatin correspond to changes in benign inflammatory dermatoses.

Example 4 . Patient D., 48 years old, N case history - 16105, MONIKI.

Entered the clinic with complaints of discoloration (redness), itching, chills, burning, tingling. It is sick since childhood, marks seasonal relapses every spring and autumn, the last 3 years without remission. Previously, she was treated with antihistamines for atopic dermatitis.

Clinical diagnosis at the time of admission: T-cell lymphoma of the skin.

Clinical data at admission: skin is congestive-cyanotic color, 100% erythroderma, marked infiltration, all groups of peripheral lymph nodes, especially the femoral and inguinal (up to 4 - 5 cm) are enlarged.

Using the proposed method, the structure of chromatin of peripheral blood lymphocytes in a patient is examined (see Example 1.).

Indicators of the structure of chromatin for the examined patient:

OD4 = 4.78724; FORM_P = 0.944441; IOD2 - 66646.9; AREA - 11071.9.

Further, for the complex estimation, the DF value = 1.206237183 was calculated.

The value of DF> 0, therefore, the revealed changes in the structure of chromatin correspond to changes in benign inflammatory dermatoses.

At the examination in the clinic in peripheral blood smears, single cells of Cesari (blasts, similar to the large cell variant) were found in the patient, but according to the biopsy conclusion - neurodermatitis (typical).

On the background of treatment with tavegil, activated charcoal, haemodez, and cleritin, the patient noted improvement in well-being, reduction of erythema and infiltration of the skin.

Thus, the conducted clinical examinations of the patient and the effectiveness of therapy confirmed the patient's benign inflammatory dermatosis (neurodermatitis).

Example 5 . Patient P., 80 years old, N case history - 9743, MONIKI.

Entered the clinic with complaints of itching, redness of the skin, chills, tingling, burning. Has been sick since 1970, previously treated with prednisolone for allergic dermatitis.

For 4 months before the present admission to the clinic, the patient was examined in the MONICA skin clinic with the alleged diagnosis: exfoliative dermatitis Wilson-Broca.

The result of the histological examination of the skin biopsy specimen: the changes correspond more to the generalized erythroderma.

Clinical diagnosis at the time of admission: allergic dermatitis.

Clinical data on admission: skin is bright red; Pronounced infiltration, 100% erythroderma, hair thinning, pronounced hyperkeratosis on the palms and soles, moderately enlarged axillary, inguinal and femoral lymph nodes.

Using the proposed method, the structure of chromatin of peripheral blood lymphocytes in a patient is examined (see Example 1.).

Indicators of the structure of chromatin for the examined patient:

OD4 - 4.10798; FORM_P - 0.820714; IOD2 - 42548.7; AREA 8391.45.

Further, for the complex estimation, the indicator DF = -0.9856234 was calculated.

The value of the indicator DF <0; Therefore, the revealed changes in the structure of chromatin correspond to changes in T-cell lymphoma of the skin.

At the examination in the clinic, changes in peripheral blood were detected (lymphocytes - 72%, Cesari cells - 15%); At a biopsy the diagnosis of a T-cell lymphoma is confirmed: Sisari syndrome, diffuse infiltrate with polymorphism, microabscesses, large multinucleate cells in the infiltrate.

The coincidence of the results of the tests performed with the proposed method and the clinical picture of the disease testifies to the specificity of the method of diagnosis of T-cell lymphomas of the skin (erythrodermic variants).

Thus, in comparison with the prototype, the proposed method of early diagnosis and differential diagnosis of T-cell lymphomas of the skin allows:

A) to detect malignant forms of lymphocytes at earlier stages, when Sesari cells are not yet detected and to establish the diagnosis before the development of severe clinical manifestations;

B) conduct differential diagnosis of erythrodermic conditions in T-cell lymphomas of the skin and benign inflammatory dermatoses;

C) reduce the traumatism of the patient with skin disease, since the blood for investigation is taken not from the vein, but from the finger.

REFERENCES

1. Orbaneja JG, Vus ES, Diar-Flores L., Huarte PS, Cytology of the micosis fungoides and Sezary syndrome. - Brit. J. Derm. 1972, 87, 2, 96-105.

2. Persina IS Clinical morphology of malignant skin lymphomas. - Author's abstract. Diss, Doct. honey. Sciences: - M. 1989.

3. Polliak A., Dialdettim, Reyes F. Surface Features of Sezary cells: a scanning Electron microscopy Study of 5 cases. - Scand, J, Haemat, 1977, 18, 3, 207 - 213.

4. Lennert K. S., Raiserbing E. Cytological and functional criteria for the classication of malignant lymphomata. - Br. J. Cancer 1975, 31, Suppl. II, 29-49.

5. Vloten WA, Schaberg A., van der Ploegm. Cytometric studies on mycosis fungoides and other cutaneous retieulosis. - Bull. Cancer 1977, 64.2, 249-258.

6. Dewald G., Spurbeck JL, Viteic MA Chromosomes in a patient with Sezary Syndrome. - Mago clin. Proc. 1974, 49, 8, 553-557.

7. Willemze R., Van. Vloten WA, Hermans J., Damstug MJM, Meifer CJLM Diagnostic criteria in Sezary syndrome: a multiparameter lymphosytes in 32 patients with erytroderma. - J. Invest. Derm. 1989, 81, 5, 392-397.

8. Hastrup N., Pallesen G., Ralfkiaer E. Use of monoclonal antibodies for the diagnosis of T-cell malignansies. Application and limitations. Leukemia / lymphoma 1990, 2, 35 - 45.

9. Pierce E. Histochemistry. - M., 1962, p. 190-192, 746.

10. Afifi A., Eisen S. Statistical analysis: a computer-based approach. Trans. With the English. - Moscow: Mir, 1982. - 488 p.

CLAIM

A method for diagnosing T-cell lymphomas of the skin (erythrodermic variants) by cytological analysis taken from a diseased biological material, characterized in that a blood smear is taken as the biological material, fixed, ribonuclease treated, stained with chro- matic alum, microdensitometer interphase chromatin of lymphocytes on Based on the obtained parameters (OD 4 ), the optical density of euchromatin, FORM_ P form factor of the nucleus; IOD 2 integral optical density of heterochromatin; AREA core area), calculate the DF value of -0.14379 × OD 4 + 0.00003 × 1OD 2 + 10.4952 × FORM_P + 0.001 × AREP 11,1241, and with a negative value of this index, T-cell lymphoma of the skin is diagnosed.

print version
Date of publication 01.04.2007гг