Modern methods of diagnosis and treatment of paroxysmal nocturnal hemoglobinuria

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

The leading link in pathogenesis is the loss of the GPI-AP (glycosyl-phosphatidylinositol anchor protein) protein on the cell surface due to a somatic mutation. This protein is an anchor, and when it is lost, some important proteins cannot attach to the membrane. Many proteins lose their ability to attach, which is used to diagnose paroxysmal nocturnal hemoglobinuria by immunophenotyping (erythrocytes CD59-, granulocytes CD16-, CD24-, monocytes CD14-). Cells with signs of absence of the studied proteins are called a PNH clone. All these proteins must interact with proteins of the complement system, in particular with C3b and C4b, destroying the enzymatic complexes of the classical and alternative complement pathways, and thereby stopping 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, cytopenic. Each patient may have one, two, or all three syndromes.

The "classical" form is the manifestation of the disease in the form of pronounced hemolysis ± thrombosis, the bone marrow in this form is hypercellular. A separate form of combination of paroxysmal nocturnal hemoglobinuria and bone marrow failure is distinguished (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%) PNH clone.

Hemolysis is largely associated with the absence of the CD59 protein (membrane inhibitor of reactive lysis (MIRL)) on the surface of red blood cells. Hemolysis in paroxysmal nocturnal hemoglobinuria is intravascular, so dark urine (hemosiderinuria) and severe weakness may appear. Laboratory tests show a decrease in haptoglobin (a physiological defense reaction during hemolysis), an increase in lactate dehydrogenase (LDH), a positive test for free hemoglobin in the urine (hemosiderinuria), a decrease in hemoglobin followed by an increase in reticulocytes, and an increase in the unbound fraction of bilirubin. The Hema test (hemolysis of red blood cells when a few drops of acid are added to a blood sample) and the sucrose test (adding sucrose activates the complement system) are used to diagnose paroxysmal nocturnal hemoglobinuria.

It is currently believed that hemolysis occurs almost constantly, but has periods of intensification. A large amount of free hemoglobin triggers a cascade of clinical manifestations. Free hemoglobin actively binds to nitric oxide (NO), leading to a violation of the regulation of smooth muscle tone, activation and aggregation of platelets (abdominal pain, dysphagia, impotence, thrombosis, pulmonary hypertension). Free hemoglobin that is not bound to haptoglobin damages the kidneys (acute tubulonecrosis, pigment nephropathy) and after several years can lead to renal failure. Dark urine in the morning is explained by 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 (increased LDH) does not contradict the diagnosis and is explained by the binding of free hemoglobin to haptoglobin and nitric oxide, reabsorption of hemoglobin in the kidneys.

Thrombosis is diagnosed in 40% of patients and is the main cause of death, most often thrombosis of the liver's own veins (Budd-Chiari syndrome) and pulmonary embolism. Thrombosis in paroxysmal nocturnal hemoglobinuria has its own characteristics: it often coincides with episodes of hemolysis and occurs despite anticoagulant therapy and a small PNH clone. The pathophysiological rationale for thrombosis includes platelet activation due to CD59 deficiency, endothelial activation, impaired fibrinolysis, formation of microparticles, and entry of phospholipids into the blood as a result of complement system activation. A number of authors point to 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 stem cells (GPI+) and cells with mutation (GPI-) coexist in the bone marrow. A small (less than 1%) PNH clone often appears in patients with aplastic anemia and myelodysplastic syndrome.

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

Treatment can be divided into supportive care, thrombosis prevention, immunosuppression, stimulation of erythropoiesis, stem cell transplantation, and treatment with biological agents. Supportive care includes red blood cell transfusions, folic acid, vitamin B12, and iron supplements. Most patients with the "classic" form of paroxysmal nocturnal hemoglobinuria are transfusion-dependent. Hemochromatosis with cardiac and hepatic involvement is rare in patients with paroxysmal nocturnal hemoglobinuria, since hemoglobin is filtered into the urine. Cases of renal hemosiderosis have been described.

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

Immunosuppression is performed with cyclosporine and antithymocyte immunoglobulin. During acute hemolysis, prednisolone is used in a short course.

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

Since 2002, the drug eculizumab, which is a biological agent, has been used worldwide. The drug is an antibody that blocks the C5 component of the complement system. Experience has shown increased survival, decreased hemolysis and thrombosis, and improved quality of life.

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Clinical case of the "classic" variant of paroxysmal nocturnal hemoglobinuria

Patient D., 29 years old. Complaints of weakness, yellow sclera, dark urine in the morning, some days - yellow but cloudy urine with an unpleasant odor. In May 2007, dark urine appeared for the first time. In September 2007, she was examined at the Hematology Research Center (HRC), Moscow. Based on the positive Hema test and sucrose test, detection of 37% (normal - 0) clone of erythrocytes with the immunophenotype CD55-/CD59- in the blood, hemosiderinuria, anemia, reticulocytosis in the blood up to 80% (normal - 0.7-1%), hyperbilirubinemia due to indirect bilirubin, the following diagnosis was established: paroxysmal nocturnal hemoglobinuria, secondary folate and iron deficiency anemia.

Hemolysis increased during pregnancy in 2008. In June 2008, at 37 weeks, a cesarean section was performed due to partial placental abruption and the risk of fetal hypoxia. The postoperative period was complicated by acute renal failure and severe hypoproteinemia. With intensive care, acute renal failure resolved on the fourth day, blood counts returned to normal, and edema syndrome was relieved. A week later, the temperature rose to 38-39°C, weakness, and chills. Metroendometritis was diagnosed. The therapy was ineffective, and an extirpation of the uterus and tubes was performed. The postoperative period was complicated by liver failure with syndromes of cholestasis, cytolysis, mesenchymal inflammation, severe hypoproteinemia, and thrombocytopenia. According to ultrasound, thrombosis of the liver's own veins and portal vein was diagnosed. Antibacterial and anticoagulant therapy, administration of hepatoprotectors, prednisolone, replacement therapy with FFP, EMOLT, and platelet concentrate were carried out.

She was re-hospitalized in the State Research Center due to thrombosis of the portal and proper veins of the liver, thrombosis of small branches of the pulmonary artery, development of infectious complications, with rapidly increasing ascites. Intensive anticoagulant therapy, antibiotic therapy led to partial recanalization of the portal vein and proper veins of the liver, a decrease in ascites was noted. Subsequently, the patient was administered low-molecular heparin - Clexane for a long time.

Currently, according to laboratory parameters, the patient still has hemolysis - a decrease in hemoglobin to 60-65 g / l (normal 120-150 g / l), reticulocytosis up to 80% (normal - 0.7-1%), an increase in the LDH level to 5608 U / l (normal - 125-243 U / l), hyperbilirubinemia up to 300 μmol / l (normal - 4-20 μmol / l). Immunophenotyping of peripheral blood - the total value of the erythrocyte PNH clone is 41% (normal - 0), granulocytes - FLAER- / CD24- 97.6% (normal - 0), monocytes - FLAER- / CD14 - 99.3% (normal - 0). Continuous replacement therapy with washed red blood cells (2-3 transfusions every 2 months), folic acid, iron preparations, vitamin B12 is carried out _. Given the very high thrombogenic risk, warfarin therapy is carried out (INR - 2.5). The patient is included in the national registry of PNH for planning therapy with eculizumab.

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, increased body temperature to 38 degrees.

The onset of the disease is gradual, about 1 year, when bruises began to appear on the body. Six months ago, bleeding gums appeared, general weakness increased. In April 2012, a decrease in hemoglobin to 50 g / l was recorded. In the Central Regional Hospital, the therapy with vitamin B ₁₂ and iron preparations did not give a positive effect. In the hematology department of the Republican Clinical Hospital - 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), an increase in LDH - 349 U / l (norm 125-243 U / l).

According to bone marrow aspiration biopsy data, there is a decrease in the megakaryocytic lineage. Immunophenotyping of peripheral blood: the total value of the erythrocyte PNH clone is 5.18%, granulocytes - FLAER-/CD24 - 69.89%, monocytes - FLAER-/CD14- 70.86%.

The patient underwent three transfusions of red blood cells. Allogeneic stem cell transplantation or biological therapy are currently being considered.

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

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