Modern methods of diagnosis and treatment of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (APG) is a rare (orphanic) disease. Mortality in paroxysmal nocturnal hemoglobinuria is about 35% within 5 years of the onset of the disease. Unfortunately, most cases remain undiagnosed. Clinical manifestations are diverse and patients can be observed with such diagnoses as aplastic anemia, unclear etiology thrombosis, hemolytic anemia, refractory anemia (myelodysplastic syndrome). The average age of patients is 30-35 years.

The leading link in the pathogenesis is the loss, due to somatic mutation, of the protein GPI-AP (glycosyl-phosphatidylinositol anchor protein) on the cell surface. This protein is an anchor, with the loss of which some important proteins can not join the membrane. The ability to join is lost by many proteins, which is used to diagnose paroxysmal nocturnal hemoglobinuria by immunophenotyping (CD59-erythrocytes, CD16-, CD24-, CD14- monocytes). Cells with signs of absence of the proteins being studied are called APG clones. All these proteins must interact with complement proteins, in particular with C3b and C4b, destroying the enzymatic complexes of the classical and alternative complement pathways, and thereby stop the complement chain reaction. The absence of the above proteins leads to the destruction of cells upon activation of the complement system.

There are three main clinical syndromes in paroxysmal nocturnal hemoglobinuria: hemolytic, thrombotic, and cytopenic. Each patient can have one, two or all three syndromes.

"Classical" form refers to manifestations of the disease in the form of pronounced hemolysis ± thrombosis, bone marrow with this form - hypercellular. Isolate a separate form of a combination of paroxysmal nocturnal hemoglobinuria and bone marrow failure (paroxysmal nocturnal hemoglobinuria + aplastic anemia, paroxysmal nocturnal hemoglobinuria + myelodysplastic syndrome), when there are no pronounced clinical manifestations, but there are indirect laboratory signs of hemolysis. Finally, there is a third subclinical form in which there are no clinical and laboratory signs of hemolysis, but there is bone marrow failure and a small (S 1%) APG clone.

Hemolysis is largely due to the absence of the CD59 protein (membrane inhibitor of reactive lysis (MIRL)) on the surface of erythrocytes. Hemolysis with paroxysmal nocturnal hemoglobinuria intravascular, therefore, dark urine (hemosiderinuria) and severe weakness may appear. The reduction of haptoglobin (the reaction of physiological protection during hemolysis), increasing lactate dehydrogenase (LDH), a positive test for free hemoglobin in the urine (hemosiderinuria), a decrease in hemoglobin with subsequent reticulocyte growth, an increase in the unbound fraction of bilirubin are recorded in laboratories. The Hema assay (hemolysis of red blood cells with the addition of several drops of acid to the blood sample) and a sucrose test (addition of sucrose activates the complement system) are used to diagnose paroxysmal nocturnal hemoglobinuria.

At present, it is believed that hemolysis flows almost constantly, but has periods of amplification. A large amount of free hemoglobin is triggered by a cascade of clinical manifestations. Free hemoglobin actively binds to nitric oxide (NO), resulting in impaired regulation of smooth muscle tone, activation and aggregation of platelets (abdominal pain, dysphagia, impotence, thrombosis, pulmonary hypertension). Free hemoglobin, not associated with haptoglobin, damages the kidneys (acute tubulonecrosis, pigmentary nephropathy) and in a few years can lead to kidney failure. Dark morning urine is due to the activation of the complement system due to respiratory acidosis during sleep. The absence of dark urine in some patients in the presence of other laboratory signs of hemolysis (an increase in LDH) does not contradict the diagnosis and is explained by the binding of free hemoglobin with haptoglobin and nitric oxide, the reabsorption of hemoglobin in the kidneys.

Thrombosis is diagnosed in 40% of patients and is the leading cause of death, most often thrombosis of the liver's own veins (Badd-Chiari syndrome) and PE. Thrombosis during paroxysmal nocturnal hemoglobinuria has features: often coincide with episodes of hemolysis and occur despite anticoagulant therapy and a small APG clone. In the pathophysiological justification of thrombosis, platelet activation is considered in view of the lack of CD59, endothelial activation, impaired fibrinolysis, microparticle formation and phospholipid entry into the blood as a result of activation of the complement system. A number of authors indicate an increase in D-dimers and abdominal pain, as the main predictors of thrombosis.

The pathogenesis of bone marrow failure syndrome in paroxysmal nocturnal hemoglobinuria is unclear. Normal bone stem cells (GPI +) and mutated cells (GPI-) coexist in the bone marrow. Often there is a small (less than 1%) APG clone in patients with aplastic anemia and myelodysplastic syndrome.

The gold standard for the diagnosis of paroxysmal nocturnal hemoglobinuria is the immunophenotyping of peripheral blood cells for the presence of APG clone. In conclusion, the study indicates the size of the APG clone in erythrocytes (CD 59-), granulocytes (CD16-, CD24-) and monocytes (CD14-). Another method of diagnosis is FLAER (fluorescently labeled inactive toxin aerolysin), a bacterial toxin aerolysin labeled with fluorescent labels, which binds to the GPI protein and initiates hemolysis. The advantage of this method is the possibility of testing all cell lines in one sample, a disadvantage is the impossibility of testing with a very low amount of granulocytes, which is observed with aplastic anemia.

Treatment can be divided into maintenance therapy, prevention of thrombosis, immunosuppression, stimulation of erythropoiesis, stem cell transplantation, treatment with biological agents. Supportive therapy includes transfusion of erythrocytes, the appointment of folic acid, vitamin B12, iron preparations. Most patients with a "classical" form of paroxysmal nocturnal hemoglobinuria depend on blood transfusions. Hemochromatosis with heart and liver damage in patients with paroxysmal nocturnal hemoglobinuria is rare, as hemoglobin is filtered into urine. Cases of hemosiderosis of the kidneys are described.

Prevention of thrombosis is carried out by warfarin and low-molecular heparin, INR should be at the level of 2.5-3.5. The risk of thrombosis does not depend on the size of the APG clone.

Immunosuppression is performed by cyclosporine and antitumocyte immunoglobulin. During acute hemolysis, prednisolone is administered in a short course.

Stem cell transplantation is the only method that gives the chance of a complete cure. Unfortunately, the complications and difficulties of donor selection, associated with allogeneic transplantation, limit the use of this method. The mortality of patients with paroxysmal nocturnal hemoglobinuria in allogeneic transplantation is 40%.

Since 2002, the world uses the drug ekulizumab, which is a biological agent. The drug is an antibody blocking the C5 component of the complement system. The experience of the application showed an increase in survival rate, a decrease in hemolysis and thromboses, an improvement in the quality of life.

[1], [2], [3], [4], [5], [6]

The clinical case of the "classical" variant of paroxysmal nocturnal hemoglobinuria

Patient D., 29 years old. Complaints of weakness, yellow color sclera, dark urine in the morning, some days - urine yellow, but cloudy, with an unpleasant odor. In May 2007, dark urine first appeared. In September 2007, she was examined in the hematological research center (SSC), Moscow. Based on the presence of a positive sample of Hem and a sucrose test, a blood clone of 37% (norm-0) of the erythrocyte clone with immunophenotype CD55- / CD59-, hemosiderinuria, anemia, reticulocytosis in the blood up to 80% (0.7-1% norm), hyperbilirubinemia due to indirect bilirubin, a diagnosis was made: paroxysmal nocturnal hemoglobinuria, secondary folio-and iron-deficiency anemia.

Hemolysis intensified against the background of pregnancy in 2008. In June 2008, at 37 weeks, a cesarean section was performed in connection with partial placental abruption and the threat of fetal hypoxia. The postoperative period was complicated by acute renal failure, severe hypoproteinemia. Against the backdrop of intensive therapy, OPN resolved on the fourth day, the blood counts returned to normal, the edematous syndrome was stopped. After a week, the temperature rises to 38-39 ° C, weakness, chills. Metroendometritis was diagnosed. Conducted therapy was ineffective, extirpation of the uterus with tubes was performed. The postoperative period was complicated by hepatic insufficiency with syndromes of cholestasis, cytolysis, mesenchymal inflammation, severe hypoproteinemia, thrombocytopenia. According to ultrasound, thrombosis of the liver's own veins and portal vein is diagnosed. Antibacterial and anticoagulant therapy, the introduction of hepatoprotectors, prednisolone, replacement therapy of FFP, EMOLT, thromboconcentrate were carried out.

She was re-hospitalized in the State Scientific Center in connection with thrombosis of the portal and own veins of the liver, thrombosis of small branches of the pulmonary artery, the development of infectious complications, with rapidly increasing ascites. Conducted intensive anticoagulant therapy, antibiotic therapy led to a partial recanalization of the portal vein and its own liver veins, a decrease in ascites was noted. Later, the patient was injected with low-molecular weight heparin-clexane for a long time.

At present, according to laboratory parameters, the patient retains hemolysis - a decrease in hemoglobin to 60-65 g / l (norm 120-150 g / l), reticulocytosis to 80% (the norm is 0.7-1%), an increase in the LDH level to 5608 U / l (norm - 125-243 E / L), hyperbilirubinemia up to 300 μmol / l (the norm is 4-20 μmol / l). Immunophenotyping of peripheral blood - the total value of erythrocyte APG clone 41% (normal - 0), granulocytes - FLAER- / CD24- 97,6% (norm-0), Monocytes - FLAER- / CD14 - 99,3% (norm-0) . Constant replacement therapy with washed red blood cells (2-3 transfusions every 2 months), folic acid, iron preparations, vitamin B _{12 is} carried out . Given a very high thrombogenic risk, therapy with warfarin (INR 2.5) is performed. The patient is entered in the national APG register for planning the treatment with the drug ekulizumab.

A clinical case of a combination of aplastic anemia and paroxysmal nocturnal hemoglobinuria

Patient E., 22 years old. Complaints of general weakness, tinnitus, bleeding gums, bruises on the body, weight loss of 3 kg, body temperature increase to 38 g.

The onset of the disease is gradual, about 1 year, when bruises on the body began to appear. Six months ago, bleeding gums joined, general weakness increased. In April 2012, there was a decrease in hemoglobin to 50 g / l. In the CRH carried out therapy with vitamin B ₁₂, iron preparations did not give a positive effect. In the hematological department of the RCB - severe anemia, Hb - 60 g / l, leukopenia 2.8x10 ⁹ / l (norm - 4.5-9x10 ⁹ / l), thrombopenia 54x10 ⁹ / l (norm - 180-320x10 ⁹ / l), increase LDG - 349 U / l (the norm is 125-243 U / l).

According to bone marrow aspiration biopsy, a decrease in the megakaryocyte germ. Immunophenotyping of peripheral blood - the total value of erythrocyte APG clone 5.18%, granulocytes - FLAER- / CD24 - 69.89%, Monocytes - FLAER- / CD14- 70.86%.

The patient was transfused three times with erythrocyte mass. At present, the possibility of allogeneic stem cell transplantation or the appointment of biological therapy is being considered.

Assistant of the Department of Hospital Therapy of KSMU Kosterina Anna Valentinovna. Modern methods of diagnosis and treatment of paroxysmal nocturnal hemoglobinuria // Practical medicine. 8 (64) December 2012 / volume 1

[7], [8], [9], [10], [11], [12], [13], [14]