RU2599021C1 - Method for prediction of course of immune thrombocytopenia following splenectomy by weight of cd4+ t-lymphocytes of spleen - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, specifically to haematology, and discloses a method for prediction of course of immune thrombocytopenia (ITP) following splenectomy by weight of CD4+ T-lymphocytes of spleen. Method is characterised by that after removal of spleen, its weight is determined, in histological sections of lymphoid organ after immunohistochemical colouring morphometrically counting number of CD4+ T-lymphocytes and their mass is calculated by formula: M=mxD/100%, where M is mass of CD4+ T-lymphocytes (g), m is mass of spleen (g), D is number of CD4+ T-cells (%), and if mass of CD4+ T-lymphocytes is less or equal to 20.5 g refractory form of immune thrombocytopenia is diagnosed, and if more than 20.5 g, favourable prognosis of disease is established.

EFFECT: use of present method enables to predict course of ITP following splenectomy and performing a differentiated approach when prescribing therapy.

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Description

The invention relates to medicine, in particular to hematology, and can be used to predict the course of immune thrombocytopenia (ITP) after splenectomy by weight of spleen CD4 + T-lymphocytes by determining the mass of lymphoid organ, morphometric estimation of the number of CD4 + T-cells in histological sections of the spleen after immunohistochemical (IHC) staining and calculating their mass.

The purpose of the invention is the development of a method for predicting the course of ITP after splenectomy by weight of splenic CD4 + T-lymphocytes.

ITP belongs to the group of autoimmune hematological diseases and is characterized by a decrease in the content of blood platelets in peripheral blood. The occurrence of the pathological process is associated with the destruction of platelets by autoantibodies with the participation of macrophages of the spleen, which indicates the central role of this organ in the pathogenesis of the disease (A. A. Maschan, A. G. Rumyantsev, L. G. Kovaleva [et al.] / Recommendations of the Russian Council experts on the diagnosis and treatment of patients with primary immune thrombocytopenia // Oncohematology. - 2010. - No. 3. - P. 36-45; F. Rodeghiero, R. Stasi, T. Gernsheimer [et al.] / Standardization of terminology, definitions and autcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group // Blood. - 2009. - Vol. 113, No 11. - P. 2386-2393 ) Splenectomy is part of the standard treatment program for patients with ITP. There is still no clear idea of the causes of refractory forms of the disease. In recent years, there have been reports of a T-cell mechanism for inhibiting activation of cell lines involving CD4 + regulatory lymphocytes, which play a leading role in the immune response in ITP (W. Bao, JB Bussel, S. Heck [et al.] / Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents // Blood. - 2010. - Vol. 116, No 22. - P. 4639-4645; BH Chong / ITP: Tregs come to the rescue // Blood. - 2010. - Vol. 116, No. 22 .-- P. 4388-4390).

There is no method for predicting the course of ITP after splenectomy by weight of splenic CD4 + T-lymphocytes.

No prototype.

An analogue is the known method for assessing spleen CD4 + and CD8 + T cells in ITP patients not responding to rituximab therapy (S. Audia, M. Samson, M. Mahevas [et al.] / Preferential splenic CD8 + T-cell activation in rituximab-nonresponder patients with immune thrombocytopenia // Blood. - 2013. - Vol. 122. - P. 2477-2486).

Differences of the proposed method from the analogue include:

1. In the claimed method, morphometrically after IHC staining of histological sections of the spleen, the number of CD4 + T-lymphocytes is counted, their mass is calculated, and ITP variants after splenectomy are predicted by the mass values of CD4 + cells.

2. In the analogue, the results of ITP treatment in patients who do not respond to rituximab therapy are evaluated cytophotometrically by the relative number (%) of CD8 + and CD8 + / CD4 + T cells in the spleen and blood; the method does not allow predicting the course of ITP.

In the process of conducting a patent information search, no sources were found discrediting the novelty of the invention.

The claimed invention was developed in the laboratory of blood pathomorphology FGBUN KNIIIGiPK FMBA of Russia in accordance with the plan of research work.

The method is as follows.

After splenectomy in patients with ITP, the mass of the removed spleen is determined. The histological posting of samples of the test material is performed in a conventional manner; the preparation of sections is carried out from paraffin blocks of the spleen, 3-5 microns thick. To identify CD4 + T-lymphocytes in the spleen, the IHC staining method is used; The reaction is carried out according to standard methods. A morphometric estimate of the number of CD4 + T-lymphocytes is performed using a light microscope with an integrated photo and video camera using ImageScope Color image analysis software, version M, with an eyepiece × 10, with a lens × 100. Studies are carried out in 20 fields of view for each sample, starting from the capsule deep into the lymphoid organ. Based on the results obtained, calculate the mass of CD4 + T-lymphocytes of the spleen according to the formula:

M = m × D / 100%,

Where

m is the mass of the spleen, g;

D is the number of CD4 + T-lymphocytes,%;

M - mass of CD4 + T-lymphocytes, g.

The studies were performed on histological sections of spleen paraffin blocks from 32 patients with ITP after splenectomy aged 19 to 74 years (12 males and 20 females). The comparison group consisted of samples of spleens taken from 20 people who did not have a history of blood system diseases. After splenectomy, the spleen mass was determined, postoperative material samples were posted, morphometrically after IH staining, the number of spleen CD4 + T lymphocytes was counted, their mass was calculated; with CD4 + T-lymphocyte mass values less than and equal to 20.5 g, the refractory form of ITP was predicted, and with more than 20.5 g, an ITP variant with a favorable course of the disease was predicted. According to the analysis results, in 15 patients after splenectomy, a refractory form of the pathological process was established and in 17 - a favorable variant of the course of the disease.

The research results are presented in examples confirming the possibility of the proposed method to predict ITP course variants after splenectomy by weight of splenic CD4 + T cells.

Example 1

Patient M, 59 years old, was admitted to the hematology department of the Federal State Budgetary Scientific Institution KNIIIGiPK FMBA of Russia on 03.04.2011. Blood test on admission: hemoglobin 154 g / l, red blood cells 5.5 × 10 ¹² / _l , white blood cells 14.4 × 10 ⁹ / _l , platelets 9 × 10 ⁹ / _L. Diagnosis at admission: ITP, severe form, poorly controlled by drugs. After 2 months, the operation was performed (June 7, 2011): splenectomy - without complications. The patient’s spleen mass (m) was 119.0 g. IHC staining of lymphocytes in histological sections of the spleen was performed according to the standard method. Using morphometric studies determined the number of CD4 + T-lymphocytes (D) - 13.4%. The mass (M) of CD4 + T cells was calculated using the previously given formula: 15.9 g. The determined value was obtained in the following calculation: M = 119.0 g × 13.4% / 100.

Conclusion: according to the results of IHC and morphometric analysis, it was found that a patient with ITP has a refractory form of the course of the disease. During dynamic observation of the patient, 2 months after splenectomy, the prognosis was confirmed by laboratory parameters (08/15/10): hemoglobin 134 g / l, red blood cells 4.89 × 10 ¹² / _l , white blood cells 10.3 × 10 ⁹ / _l , platelets 75 × 10 ⁹ / _l . The patient still needs supportive care.

Example 2

Patient B., 53 years old, was admitted to the hematology department of the Federal State Budget Scientific Institution KNIIGiPK FMBA of Russia on October 30, 2009. A blood test at admission: hemoglobin 148 g / l, red blood cells 5.69 × 10 ¹² / _l , white blood cells 6.9 × 10 ⁹ / _l platelets 26 × 10 ⁹ / _L. Diagnosis at admission: ITP, severe form, poorly controlled by drugs. After 14 months, the operation was performed (12/10/2010): splenectomy - without complications. The patient’s spleen mass (m) was 155.0 g. IHC staining of lymphocytes in histological sections of the spleen was performed according to the standard method. Morphometrically determined the number of CD4 + T-lymphocytes (D) - 15.7%. The mass (M) of CD4 + T cells was calculated using the above formula: 24.3 g. The determined value was obtained in the following calculation: M = 155.0 g × 15.7% / 100.

Conclusion: according to the results of IHC and morphometric analysis, it was found that the ITP patient has a favorable variant of the course of the disease. During dynamic observation of the patient, the prognosis was confirmed by laboratory parameters, the values of which were within normal limits for 5 months after splenectomy (04/26/11): hemoglobin 138 g / l, red blood cells 5.01 × 10 ¹² / _l , white blood cells 10.8 × 10 ⁹ / _L , platelets 204 × 10 ⁹ / _L. Currently, the patient does not need maintenance therapy.

The claimed invention enables the mass of CD4 + T-lymphocytes of the spleen to predict the course of ITP after splenectomy. Lower mass values of CD4 + T-lymphocytes indicate a refractory form of the pathological process, on the contrary, higher in comparison with the comparison group indicates a favorable course of the disease. The proposed method is informative, can be used in hematological and pathomorphological departments, morphological and histological laboratories as an additional criterion in predicting the course of ITP and for a differentiated approach when prescribing therapy.

Claims (1)

A method for predicting the course of immune thrombocytopenia after splenectomy by weight of spleen CD4 + T lymphocytes, characterized in that after removal of the spleen its mass is determined, in histological sections of the lymphoid organ after immunohistochemical staining, the number of CD4 + T lymphocytes is morphometrically calculated and their mass is calculated by the formula: M = = m × D / 100%, where M is the mass of CD4 + T-lymphocytes (g), m is the mass of the spleen (g), D is the number of CD4 + T-cells (%), and when the mass of CD4 + T-lymphocytes is less than and equal to 20 5 g state a refractory form moon thrombocytopenia, and with more than 20.5 g establish a favorable prognosis of the course of the disease.