RU2469322C1 - Method for prediction of risk of developing inhibitory form of haemophilia a - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: blood 5 ml stabilised by heparin or EDTA is taken from a patient; a ficoll-verografin density lymphocyte suspension is recovered, and patient's HLA-phenotype (A and B loci) is described by microlymphotoxic test with a kit of histotyping serums. If the antigen HLA-A28 is found in the patient's phenotype, a higher risk of production of factor VIII preparation inhibitors is predicted. If the antigen HLA-B8 is found in the patient, a lower risk of developing complication of replacement therapy is predicted.

EFFECT: use of the declared method enables higher effectiveness of prediction of a risk of developing inhibitory form of haemophilia A.

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Description

The invention relates to medicine, namely to hematology, and for the determination of risk factors for the production of inhibitors in hemophilia A patients receiving replacement therapy with factor VIII drugs.

Hemophilia is a hereditary disease of the hemostatic system, characterized by a deficiency of factor VIII (hemophilia A) or IX (hemophilia B). The prevalence of hemophilia A is 1: 10000, hemophilia B is 1: 30,000-50000 males. To date, genes have been mapped that control the synthesis of factors VIII and IX and are responsible for the development of hemophilia. The severity of the clinical course of the disease depends on the level of the factor. About 70% of patients suffer from severe and moderate forms of hemophilia.

The diagnosis of hemophilia is established on the basis of the corresponding clinical manifestations, genealogical data, the results of a study of hemostasis indicators: activated partial (partial) thromboplastin time and the activity of factors VIII and IX. Hemophilia patients are assisted by hematologists. Substitution therapy is carried out by intravenous administration of coagulation factors. Among the most serious complications of substitution therapy is the appearance of inhibitors (antibodies) to coagulation factors. Antibodies to factor VIII are produced much more often (in 10-35% of patients) than to factor IX (in 3-5%). The appearance of antibodies is accompanied by rapid inactivation of the exogenous factor, as a result, the effectiveness of treatment is reduced and bleeding may become uncontrolled. The severity of the disease is considered a risk factor for the development of inhibition; other risk factors have not been established.

A large team of authors developed a protocol for the management of hemophilia patients, which presents the requirements for diagnosis, prevention, and organization of medical care for this category of patients [1]. However, the protocol does not contain any criteria for determining the susceptibility of patients to the production of inhibitors. Meanwhile, the knowledge of such criteria would make it possible to single out among patients patients with an increased risk of inhibition who need systematic monitoring of the appearance of antibodies and their timely elimination.

The aim of the invention is to develop a method for predicting the development of an inhibitory form of hemophilia A in patients receiving replacement therapy with factor VIII drugs.

According to current data, the development of the cascade of the immune response goes through a number of stages: presentation of the antigen (processing), amplification of T-helpers with different functional activity, isolation of cytokines for various purposes, signaling to effector cells that complete the immune response either by antibody formation or cytotoxic lysis. Most often both of these processes are combined, but one type of answer is dominant, the other auxiliary. Moreover, at all stages of the immune cascade, the participation of HLA system products is mandatory [2-5]. In this regard, it is logical to assume that such a feature of the immune response as the production of antibodies to coagulation factors in hemophilia patients can be associated with specific antigens of the HLA complex. Based on this assumption, we studied the distribution pattern of HLA antigens in 107 patients with hemophilia A, of which 49 were diagnosed with an inhibitory form of the disease. Testing of HLA antigens of class I (loci A and B) was carried out using a standard lymphocytotoxic test with a set of typing sera produced by ZAO Gisans (St. Petersburg), registration certificate No. FSR 2008/02431 dated April 9, 2008). Identified 8 antigens of locus A and 16 antigens of locus B. Lymphocytes for the test were isolated from venous heparinized blood of patients by gradient centrifugation using a solution of ficoll-verographin; the test was performed on Terasaki microplates.

The frequency of occurrence of the studied antigens was determined as the percentage of individuals carrying the antigen to the total number examined in the group. To establish significant differences in the nature of the distribution of antigens in the compared groups, the agreement criterion X ^{2 was} calculated.

To identify features in the nature of the distribution of HLA antigens in the examined patients, we compared their incidence with similar parameters in the comparison group, which consisted of 795 healthy individuals (blood donors and its components who underwent medical examination). The results are presented in the table.

A quantitative assessment of the association between HLA antigens and the development of an inhibitory form of hemophilia was established by calculating the relative risk index (RR) according to the formula [2, 6]:

where f _n is the number of carriers of a particular antigen among patients,

f _k is the number of carriers of the same antigen in the healthy group;

f _n and f _k are expressed in decimal fractions.

At RR> 2.0, a positive association between the antigen and the disease (predisposition) is established, if RR <1.0, then this antigen is considered as a protective genetic trait.

As can be seen from the data presented in the table, significant differences in the frequency of occurrence of HLA antigens were revealed for two specificities: A28 was more often identified in the general group of patients (X ² = 4.5; RR = 2.3) and in patients with an inhibitory form (X ² = 6.5; RR = 3.3). Antigen B8 was significantly less frequently tested in the lymphocytes of patients with inhibitors (X ² = 4.1; RR = 0.14). It should be noted that the increase in the incidence of A28 antigen in the general group of examined patients is explained by the presence of 49 patients with an inhibitory form among them.

Table Frequency of HLA - Class I Antigens in Hemophilia Patients HLA antigens Detection rate,% Healthy
n = 795 Hemophilia patients general group
n = 107 with inhibitors
n = 49 no inhibitors
n = 58 A1 22.9 24.3 18,4 29.3 A2 47.0 50,5 53.1 48.3 A3 33.7 32,7 30.6 34.5 A9 27,2 19.6 26.5 13.8 A10 16.3 23.3 24.5 22.4 A11 13.6 10.3 8.2 12.1 A19 13.1 14.0 10,2 17,2 A28 4.8 10.3 * 14.3 ** 6.9 AT 5 11.6 12.1 14.3 10.3 AT 7 26.2 28.9 30.6 27.6 AT 8 12.8 8.4 2.0 * 13.8 AT 12 15,2 17.7 13,4 17,2 B13 10.3 11,2 14.3 8.6 B14 4.8 4.7 8.2 1.7 B15 11.5 11,2 12,2 10.3 B16 7.7 12.1 16.3 8.6 B17 7.0 10.3 10,2 10.3 B18 11.3 14.0 10,2 17,2 AT 21 4.0 6.5 8.2 5.2 B22 5,4 1.9 0,0 3.4 B27 11.0 13.1 12,2 13.8 B35 24.8 22.4 26.5 19.0 B40 12,2 9.3 6.1 12.1 B41 2,4 3,7 6.1 1.7 Note: * differences are significant at p <0.05; ** - p <0.01.

Thus, according to the results obtained, the risk of inhibitors is associated with the presence of the HLA-A28 antigen in the phenotype of the patient: it is 3.3 times higher than in the absence thereof. HLA-B8 antigen showed a protective value, which reduces the indicated risk (RR = 0.14).

Based on the presented data, it was concluded that HLA-A28 and HLA-B8 antigens are a marker of the genetic predisposition to inhibitor production in hemophilia patients, the first of which increases the risk of developing this complication, while the second reduces it.

The developed method for determining the predisposition of hemophilia A patients to the production of inhibitors allows us to identify a group of patients with an increased risk of developing this serious complication who need systematic monitoring of the appearance of antibodies for timely treatment of necessary therapeutic measures (plasmapheresis, extracorporeal immunosorption, induction of immune tolerance, etc.). The method will find application in clinical diagnostic laboratories of specialized medical institutions that provide medical care to patients with hemophilia; The proposed research method is technically and economically affordable.

Clinical examples

1. Patient A.D., born in 1979

Diagnosis: Hemophilia A, moderate form; recurrent hemarthrosis of the left shoulder joint; multiple arthropathy.

Date of receipt: November 2, 2009

Date of discharge: November 19, 2009

Indicators of coagulation hemostasis from 11/03/2009: fibrinogen 2.75 g / l; prothrombin index 97%; APTV index 2.82; F. VIII 2%; thrombin time 16 s .; RFMC 35 μg / ml; fibrinolytic activity (according to Kovarzhik) 11 min; antithrombin III 100%; inhibitors to f.VIII 1,15 units / ml.

Treatment: Immunate 1000 units, prednisone according to the scheme, plasmapheresis (1 procedure).

Recommendations are given: prophylactically transfusion of 2000 units. factor VIII 3 times a week.

HLA - phenotype: A26, 28; At 14, 15.

2. Patient P. P. N., born in 1988

Diagnosis: hemophilia A, moderate form; gingival bleeding.

Date of receipt 02.08.2010

Date of discharge: August 6, 2010

Indicators of coagulation hemostasis: fibrinogen 3.5 g / l; F. VIII 2%; prothrombin index 82%; APTV index 2.21; thrombin time 17 s; RFMC 50 μg / ml; fibrinolytic activity 9 min, antithrombin III 125%; inhibitors to f.VIII 1.6 units / ml.

Treatment: concentrate F.VIII 6000 units.

Recommendations are given: prophylactically transfusion of 2000 units. factor VIII 3 times a week.

HLA phenotype: A11, 28; B15, 35; C4

3. Patient M.A.N., born in 1956

Diagnosis: hemophilia A, moderate form, retroperitoneal hematoma on the right, posthemorrhagic anemia.

Date of receipt: September 10, 2008

Date of discharge: October 13, 2008

Indicators of coagulation hemostasis: fibrinogen 3.5 g / l; F. VIII 2%; prothrombin index 100%; APTV index 1.8; thrombin time 17 s .; RFMC 75 μg / ml; antithrombin III 100%; fibrinolytic activity for 20 min, inhibitors to f.VIII were not found.

Treatment: concentrate f.VIII 48500 units, plasmapheresis (4 procedures).

Recommendations are given: prophylactically transfusion of 2000 units. factor VIII 3 times a week.

HLA - phenotype: A1, 3; B8, 35; C4.

Thus, in patients with the presence of antigen A28 in the HLA phenotype (examples 1 and 2), an inhibitory form of hemophilia A developed, and in a patient who had antigen B 8 in the phenotype, despite the high therapeutic dose of the factor VIII concentrate, inhibitors did not appear.

Literature

1. Andreev Yu.N., Barkagan Z.S., Buevich E.I. et al. von Willebrand disease. Hemophilia // Management protocols. M., 2009 .-- 197 p.

2. Zaretskaya Yu.M. Clinical immunogenetics. - M., 1983. 208 p.

3. Khaitov P.M., Alekseev L.P. The physiological role of the main human histocompatibility complex // Immunology. - 2001. - No. 3. - S. 4-12.

4. Khaitov P.M., Alekseev L.P. HLA genomics: new possibilities of human molecular genetics in diagnostics and therapy. - Molek. the medicine. - 2003. - No. 1. - S. 17-31.

5. Dausset Y., Contu L. The MNC and immune response in man // Immunol. - 80.4 ^th Int. Congr. Immunol., Paris, Yuli, 1980. - P.513-529.

6. Sveygaard A., Ryder L. P. HLA and disease associations: detecting the strongest associations // Tissue Antigens. - 1994 .-- V.43. - P.18-27.

Claims (1)

A method for predicting the risk of developing an inhibitory form of hemophilia A, including, along with the study of hemostasis indicators, typing of HLA class I antigens and in the presence of a sick HLA-A28 antigen in the phenotype of the patient, an increased risk of inhibitors to factor VIII drugs is predicted, and risk is observed in carriers of HLA-B8 antigen the development of this complication of replacement therapy is reduced.