PNG blood disease. Paroxysmal nocturnal hemoglobinuria in adults

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare (orphan) disease with a varied clinical picture. Loss of the GPI-AP protein, due to somatic mutation on the cell surface, is a leading link in pathogenesis. Hemolysis, thrombosis and cytopenias are characteristic clinical manifestations. The gold standard for diagnosis is flow cytometry. Stem cell transplantation and the biological agent eculizumab are the most modern methods treatment.

Modern methods of diagnosis and treatment of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (APG) is a rare (orphan) disease with varied clinical presentation. The loss of protein GPI-AP, due to somatic mutation on the cell surface, is the leading players in the pathogenesis. Hemolysis, thrombosis and cytopenia are characteristic symptoms. The gold standard of diagnosis is flow cytometry. Transplantation of stem cells and biological agent ekulizumab are the most modern methods of treatment.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare (orphan) disease. The mortality rate for PNH is about 35% within 5 years of 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, due to somatic mutation, of the GPI-AP protein (glycosyl-phosphatidylinositol anchor protein) on the cell surface. This protein is an anchor, if lost, some important proteins cannot attach to the membrane. Many proteins lose their ability to join, which is used to diagnose PNH by immunophenotyping (erythrocytes CD59 -, granulocytes CD16 -, CD24 -, monocytes CD14 -). Cells with signs of the absence of the studied proteins are called 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 chain reaction complement. The absence of the above proteins leads to cell destruction when the complement system is activated.

There are three main clinical syndrome with PNH: 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 severe hemolysis ± thrombosis; the bone marrow in this form is hypercellular. There is a separate form of combination of PNH and insufficiency bone marrow(PNH + aplastic anemia, PNH + 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 (≤ 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 PNH is intravascular, so dark urine (hemosiderinuria) and severe weakness may appear. Laboratory tests indicate 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 with a subsequent increase in reticulocytes, and an increase in the unbound bilirubin fraction. The Hem test (hemolysis of red blood cells by adding a few drops of acid to a blood sample) and the sucrose test (the addition of sucrose activates the complement system) are used to diagnose PNH.

It is currently believed that hemolysis occurs almost constantly, but has periods of intensification. A large number of free hemoglobin triggers a cascade of clinical manifestations. Free hemoglobin actively binds to nitric oxide (NO), leading to impaired regulation of smooth muscle tone, platelet activation and aggregation (abdominal pain, dysphagia, impotence, thrombosis, pulmonary hypertension). Free hemoglobin that is not bound to haptoglobin damages the kidneys (acute tubulonecrosis, pigmentary nephropathy) and after a few years can lead to renal failure. Dark urine in the morning is due to 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; thrombosis of the liver's own veins (Budd-Chiari syndrome) and pulmonary embolism are more common. Thrombosis in PNH has its own characteristics: it often coincides with episodes of hemolysis and occurs despite anticoagulant therapy and a small PNH clone. The pathophysiological basis for thrombosis discusses platelet activation due to CD59 deficiency, endothelial activation, impaired fibrinolysis, microparticle formation, and the release of phospholipids into the blood as a result of activation of the complement system. 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 PNH is unclear. In the bone marrow, normal stem cells (GPI+) and cells with a mutation (GPI-) coexist. The appearance of a small (less than 1%) PNH clone is often observed in patients with aplastic anemia and myelodysplastic syndrome.

The gold standard for diagnosing PNH is immunophenotyping of peripheral blood cells for the presence of the PNH clone. The study conclusion 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 tags 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 maintenance therapy, thrombosis prevention, immunosuppression, stimulation of erythropoiesis, stem cell transplantation, and treatment with biological agents. Maintenance therapy includes red blood cell transfusions, administration of folic acid, vitamin B12, and iron supplements. Most patients with the “classic” form of PNH are dependent on blood transfusions. Hemochromatosis, with damage to the heart and liver, is rarely observed in patients with PNH, since hemoglobin is filtered into the urine. Cases of hemosiderosis of the kidneys 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 carried out 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 cure. Unfortunately, complications and difficulties in donor selection associated with allogeneic transplantation limit the use of this method. The mortality rate of patients with PNH after 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 with use has shown increased survival, decreased hemolysis and thrombosis, and improved quality of life. .

Clinical case of the “classic” variant of PNH.

Patient D., 29 years old. Complaints of weakness, yellow sclera, dark urine in the morning, some days the urine is yellow, but cloudy, 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 (HSC), Moscow. Based on the presence of a positive Hem test and sucrose test, detection in the blood of 37% (norm - 0) of a clone of erythrocytes with the immunophenotype CD55-/CD59-, hemosiderinuria, anemia, reticulocytosis in the blood up to 80% (norm - 0.7-1%), hyperbilirubinemia Due to indirect bilirubin, a diagnosis was made: PNH, secondary folate and iron deficiency anemia.

Hemolysis intensified during pregnancy in 2008. In June 2008, at 37 weeks, a C-section due to partial placental abruption and the threat of fetal hypoxia. Postoperative period complicated by acute renal failure and severe hypoproteinemia. On the background intensive care, acute renal failure resolved on the fourth day, blood counts returned to normal, and the edema syndrome was relieved. A week later, the temperature rises to 38-39 ° C, weakness, chills. A diagnosis of metroendometritis was made. The therapy was ineffective, 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 data, thrombosis of the native veins of the liver and portal vein was diagnosed. Antibacterial and anticoagulant therapy, administration of hepatoprotectors, prednisolone, replacement therapy FFP, EMOLT, thromboconcentrate.

She was readmitted to the State Research Center due to thrombosis of the portal and native veins of the liver, thrombosis of small branches pulmonary artery, development of infectious complications, with rapidly growing ascites. Intensive anticoagulant therapy and antibiotic therapy led to partial recanalization of the portal vein and the liver's own veins, and a decrease in ascites was noted. Subsequently, the patient was administered low molecular weight heparin – Clexane – for a long time.

Currently, according to laboratory indicators, the patient continues to have 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 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 is carried out with washed red blood cells (2-3 transfusions every 2 months), folic acid, iron supplements, and vitamin B12. Given the very high thrombogenic risk, warfarin therapy is carried out (INR – 2.5). The patient was entered into the national register of PNG for planning treatment with eculizumab.

Clinical case of a combination of aplastic anemia and PNH.

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, around 1 year, when bruises begin to appear on the body. Six months ago, bleeding gums began and general weakness increased. In April 2012, a decrease in hemoglobin to 50 g/l was registered. In the Central District Hospital, therapy with vitamin B12 and iron supplements did not produce a positive effect. In the hematology department of the Republican Clinical Hospital - severe anemia, HB - 60 g/l, leukopenia 2.8 × 10 9 / l (normal - 4.5-9 × 10 9 / l), thrombopenia 54 × 10 9 / l (normal - 180-320 × 10 9 /l), increase in LDH - 349 U/l (normal 125-243 U/l).

According to aspiration biopsy bone marrow reduction of megakaryocyte 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 received red blood cell transfusions three times. The possibility of allogeneic stem cell transplantation or biological therapy is currently being considered.

A.V. Kosterina, A.R. Akhmadeev, M.T. Savinova

Kazan State Medical University

Republican clinical Hospital Ministry of Health of the Republic of Tatarstan, Kazan

Kosterina Anna Valentinovna – assistant of the department hospital therapy KSMU

Literature:

1. Luzzatto L. Paroxysmal nocturnal hemoglobinuria. Hematology 2000 // American Society of Hematology Education Program. - 2000. - R. 28-38.

2. Parker C., Omine M., Richards S. et. al. for the International PNH Interest Group. Diagnosis and management of paroxysmal nocturnal hemoglobinuria // Blood. - 2005. - Vol. 106, No. 12. - R. 3699-709.

3. Hillman P., Lewis S.M., Bessler M., Luzzatto L., Dacie J.V. Natural history of paroxysmal nocturnal hemoglobinuria // N. Engl. J. Med. - 1995. - Vol. 333, No. 19. - R. 1253-8.

4. Brodsky R.A., Mukhina G.L., Li S. et. al. Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using fluorescent aerolysin // Am. J. Clin. Pathol. - 2000. - Vol. 114, No. 3. - R. 459-66.

5. Hall C., Richards S., Hillman P. Primary prophylaxis with warfarin prevents thrombosis in paroxysmal nocturnal hemoglobinuria (PNH) // Blood. - 2003. - Vol. 102, No. 10. - R. 3587-91.

6. Kelly R.J., Hill A., Arnold L.M. et. al. Long-term treatment with eculizumab in paroxysmal nocturnal hemoglobinuria: sustained efficacy and improved survival // Blood. - 2011. - Vol. 117, No. 25. - R. 6786-92.

Paroxysmal nocturnal hemoglobinuria, also known as Strübing-Marchiafava disease, Marchiafava-Micheli disease, is a rare disease, a progressive blood pathology, life-threatening patient. It is one of the types of acquired hemolytic anemia caused by disturbances in the structure of erythrocyte membranes. Defective cells are subject to premature decay (hemolysis) that occurs inside the blood vessels. The disease is genetic in nature, but is not considered inherited.

The incidence is 2 cases per 1 million people. The incidence is 1.3 cases per million people per year. It predominantly manifests itself in people aged 25-45 years; no dependence of incidence on gender and race has been identified. There are isolated cases of the disease in children and adolescents.

Important: average age detection of the disease - 35 years.

Causes of the disease

The causes and risk factors for developing the disease are unknown. It has been established that the pathology is caused by a mutation in the PIG-A gene, located in the short arm of the X chromosome. The mutagenic factor has not yet been identified. In 30% of cases of nocturnal paroxysmal hemoglobinuria, there is a connection with another blood disease - aplastic anemia.

The formation, development and maturation of blood cells (hematopoiesis) occurs in the red bone marrow. All specialized blood cells are formed from so-called stem, unspecialized cells that have retained the ability to divide. Formed as a result of successive divisions and transformations, mature blood cells enter the bloodstream.

A mutation in the PIG-A gene even in a single cell leads to the development of PNH. Damage to the gene also changes the activity of cells in the processes of maintaining bone marrow volume; mutant cells multiply more actively than normal ones. In the hematopoietic tissue, a population of cells producing defective blood cells is quickly formed. In this case, the mutant clone does not belong to malignant formations and may disappear spontaneously. The most active replacement of normal bone marrow cells with mutant ones occurs in the processes of restoration of bone marrow tissue after significant damage caused, in particular, by aplastic anemia.

Damage to the PIG-A gene leads to disruptions in the synthesis of signaling proteins that protect body cells from the effects of the complement system. The complement system is specific blood plasma proteins that provide general immune protection. These proteins bind to damaged red blood cells and melt them, and the released hemoglobin mixes with the blood plasma.

Classification

Based on available data on the causes and characteristics pathological changes There are several forms of paroxysmal nocturnal hemoglobinuria:

1. Subclinical.
2. Classic.
3. Associated with hematopoiesis disorders.

The subclinical form of the disease is often preceded by aplastic anemia. Clinical manifestations there is no pathology, but the presence of a small number of defective blood cells is detected only by laboratory tests.

On a note. There is an opinion that PNH is a more complex disease, the first stage of which is aplastic anemia.

The classic form occurs with typical symptoms; populations of defective red blood cells, platelets and some types of leukocytes are present in the patient’s blood. Laboratory methods studies confirm intravascular destruction of pathologically altered cells, hematopoiesis disorders are not detected.

After suffering from diseases leading to hematopoietic insufficiency, a third form of pathology develops. Expressed clinical picture and intravascular lysis of erythrocytes develop against the background of bone marrow lesions.

There is an alternative classification, according to which there are:

1. Actually PNH, idiopathic.
2. Developing as a concomitant syndrome with other pathologies.
3. Developing as a consequence of bone marrow hypoplasia.

The severity of the disease in different cases is not always related to the number of defective red blood cells. Both subclinical cases with a content of modified cells approaching 90%, and extremely severe cases with replacement of 10% of the normal population, have been described.

Development of the disease

It is currently known that in the blood of patients with paroxysmal nocturnal hemoglobinuria, three types of erythrocytes with different sensitivities to destruction by the complement system may be present. In addition to normal cells, red blood cells circulate in the bloodstream, the sensitivity of which is several times higher than normal. In the blood of patients diagnosed with Marchiafava-Micheli disease, cells were found whose sensitivity to complement was 3-5 and 15-25 times higher than normal.

Pathological changes also affect other blood cells, namely platelets and granulocytes. At the height of the disease, patients experience pancytopenia - an insufficient number of blood cells of different types.

The severity of the disease depends on the ratio between the populations of healthy and defective blood cells. The maximum content of red blood cells that are hypersensitive to complement-dependent hemolysis is achieved within 2-3 years from the moment of mutation. At this time, the first typical symptoms of the disease appear.

The pathology usually develops gradually; acute crisis onset is rare. Exacerbations occur against the background of menstruation, severe stress, acute viral diseases, surgical intervention, treatment with certain drugs (in particular, iron-containing ones). Sometimes the disease worsens when eating certain foods or for no obvious reason.

There is evidence of manifestations of Marchiafava-Micheli disease due to radiation exposure.

The dissolution of blood cells to varying degrees in patients with established paroxysmal nocturnal hemoglobinuria occurs constantly. Periods of moderate progression are interspersed with hemolytic crises, massive destruction of red blood cells, which leads to a sharp deterioration in the patient’s condition.

Outside of a crisis, patients are concerned about manifestations of moderate general hypoxia, such as shortness of breath, attacks of arrhythmia, general weakness, and deteriorating tolerance physical activity. During a crisis, abdominal pain appears, localized mainly in the navel area and in the lower back. Urine turns black, the darkest portion is in the morning. The reasons for this phenomenon have not yet been definitively established. With PNH, a slight pastiness of the face develops, and yellowness of the skin and sclera is noticeable.

On a note! A typical symptom of the disease is stained urine. In about half of known cases, the disease does not manifest itself.

In the periods between crises, patients may experience:

• anemia;
• tendency to thrombosis;
• liver enlargement;
• manifestations of myocardial dystrophy;
• tendency to inflammation of infectious origin.

When blood cells are destroyed, substances that increase clotting are released, which causes thrombosis. Blood clots may form in the vessels of the liver and kidneys; coronary and cerebral vessels are also susceptible to damage, which can lead to death. Thrombosis localized in the liver vessels leads to an increase in the size of the organ. Violations of intrahepatic blood flow entail dystrophic changes fabrics. When the portal vein system or splenic veins are blocked, splenomegaly develops. Disorders of nitrogen metabolism are accompanied by dysfunction of smooth muscles; some patients complain of difficulty swallowing, spasms of the esophagus, and erectile dysfunction is possible in men.

Important! Thrombotic complications in PNH predominantly affect the veins; arterial thrombosis rarely develops.

Video - Paroxysmal nocturnal hemoglobinuria

Mechanisms of development of complications of PNH

Hemolytic crisis is manifested by the following symptoms:

• acute abdominal pain caused by multiple thrombosis of small mesenteric veins;
• increased jaundice;
• pain in the lumbar region;
• lowering blood pressure;
• increased body temperature;
• staining urine black or dark brown.

In rare cases, a “hemolytic kidney” develops, a specific transient form of renal failure accompanied by acute anuria. Due to impaired excretory function, nitrogen-containing organic compounds accumulate in the blood, which are the end products of protein breakdown, and azotemia develops. After the patient recovers from the crisis, the content of formed elements in the blood is gradually restored, jaundice and manifestations of anemia partially fade away.

The most common course of the disease is crisis, interspersed with periods of stable, satisfactory condition. In some patients, the periods between crises are very short, insufficient to restore blood composition. Such patients develop persistent anemia. There is also a variant of the course with an acute onset and frequent crises. Over time, crises become less frequent. In especially severe cases, death is possible, which is caused by acute renal failure or thrombosis of blood vessels supplying the heart or brain.

Important! No daily patterns in the development of hemolytic crises have been identified.

In rare cases, the disease can have a long-term quiet course; isolated cases of recovery have been described.

Diagnostics

On early stages Diagnosis of the disease is difficult due to the manifestation of scattered nonspecific symptoms. Diagnosis sometimes requires several months of observation. The classic symptom - specific staining of urine - appears during crises and not in all patients. Reasons to suspect Marchiafava-Miceli disease are:

• iron deficiency of unknown etiology;
• thrombosis, headaches, attacks of pain in the lower back and abdomen for no apparent reason;
• hemolytic anemia of unknown origin;
• melting of blood cells, accompanied by pancytopenia;
• hemolytic complications associated with transfusion of fresh donor blood.

In the diagnostic process, it is important to establish the fact of chronic intravascular breakdown of red blood cells and identify specific serological signs of PNH.

In a complex of studies, if nocturnal paroxysmal hemoglobinuria is suspected, in addition to general urine and blood tests, the following are carried out:

• determination of hemoglobin and haptoglobin content in the blood;
• immunophenotyping by flow cytometry to identify defective cell populations;
• serological tests, in particular the Coombs test.

Required differential diagnosis with hemoglobinuria and anemia of other etiologies, in particular, autoimmune hemolytic anemia should be excluded. General symptoms are anemia, jaundice, increased bilirubin levels in the blood. Enlargement of the liver and/or spleen is not observed in all patients

Signs	Autoimmune hemolytic anemia	PNG
Coombs test	+	-
Increased content of free hemoglobin in blood plasma	-	+
Hartmann test (sucrose)	-	+
Hem's test (acidic)	-	+
Hemosiderin in urine	-	+
Thrombosis	±	+
Hepatomegaly	±	±
Splenomegaly	±	±

The results of the Hartmann and Hem test are specific for PNH and are the most important diagnostic signs.

Treatment

Relief of a hemolytic crisis is carried out by repeated transfusions of red blood cells, thawed or previously washed many times. It is believed that at least 5 transfusions are needed to achieve a lasting result, however, the number of transfusions may differ from the average and is determined by the severity of the patient’s condition.

Attention! Blood cannot be transfused to such patients without prior preparation. Transfusion of donor blood aggravates the crisis.

For symptomatic elimination of hemolysis, patients can be prescribed Nerobol, but relapses are possible after discontinuation of the drug.

Additionally, folic acid, iron, and hepatoprotectors are prescribed. When thrombosis develops, direct-acting anticoagulants and heparin are used.

In extremely rare cases, the patient is indicated for splenectomy - removal of the spleen.

All of these measures are supportive; they alleviate the patient’s condition, but do not eliminate the population of mutant cells.

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RCHR (Republican Center for Health Development of the Ministry of Health of the Republic of Kazakhstan)
Version: Clinical protocols Ministry of Health of the Republic of Kazakhstan - 2015

Paroxysmal nocturnal hemoglobinuria [marchiafava-micheli] (D59.5)

Oncohematology

general information

Short description

Recommended
Expert advice
RSE at the RVC "Republican Center"
healthcare development"
Ministry of Health
And social development
Republic of Kazakhstan
dated July 9, 2015
Protocol No. 6

Classification

Clinical classification:

There are 3 main forms of PNH.
1. Classic shape characterized by clinical and laboratory signs of intravascular hemolysis without signs of other diseases associated with bone marrow failure (aplastic anemia (AA), myelodysplastic syndrome (MDS), idiopathic myelofibrosis).
2. PNH diagnosed in patients with AA (AA/PNG), MDS (MDS/PNG) and extremely rarely with myelofibrosis (idiopathic myelofibrosis/PNH), when in these diseases there are clinical and/or laboratory signs of intravascular hemolysis, and a clone of cells with the PNH phenotype is determined in the peripheral blood.
3. Subclinical form diseases ( AA/sPNH, MDS/sPNH, idiopathic myelofibrosis/sPNH), diagnosed in patients without clinical and laboratory signs of hemolysis, but in the presence of a minor clone of cells with the PNH phenotype (usually<1 %). Следует отметить, что субклиническое течение ПНГ может отмечаться и при большем размере клона.

Isolation of the subclinical form of PNH has no independent clinical significance, but is necessary to ensure monitoring of such patients due to the likelihood of an increase in the size of the clone and progression of hemolysis, which can dominate among the clinical manifestations and require appropriate therapy.
Taking into account the fact that the subclinical form of PNH in AA and/or MDS has no independent clinical significance.

Classic PNG shape.
Patients with classic PNH typically have severe intravascular hemolysis with increased serum lactate dehydrogenase (LDH) levels, reticulocytosis, and decreased haptoglobin levels. With this variant of PNH there are no definitive morphological signs of other bone marrow pathologies (AA, MDS, myelofibrosis) and karyotype abnormalities are not characteristic

PNH against the background of bone marrow failure syndromes (AA/PNH, MDS/PNH).
In patients with AA/PNH and MDS/PNH, clinical and laboratory signs of intravascular hemolysis are diagnosed. At different stages of the development of the disease, symptoms of bone marrow failure or intravascular hemolysis may prevail, and in some cases there is a combination of both. Despite the fact that in patients with a small PNH clone, the disease usually occurs with minimal symptoms and only laboratory signs of intravascular hemolysis are noted, monitoring is necessary (twice a year). This is due to the fact that over time, expansion of the clone is possible with the development of severe hemolysis and a high risk of thrombotic complications.

Subclinical form of PNH (AA/sPNH, MDS/sPNH).
Patients with subclinical PNH do not have any clinical or laboratory signs of hemolysis. Small populations of GPIAP-deficient cells can only be detected using highly sensitive flow cytometry. The subclinical form of PNH can be diagnosed against the background of diseases characterized by impaired bone marrow function, mainly AA and MDS. It is very important to carefully monitor these patients to identify signs of hemolysis and clonal expansion, since 15-17% of patients with AA / subclinical PNH have Over time, the hemolytic form of AA/PNH develops.

Diagnostics

List of basic and additional diagnostic measures:
Basic (mandatory) diagnostic examinations performed on an outpatient basis:
· general blood test (counting reticulocytes in a smear);
· immunophenotyping of peripheral blood to determine the percentage of PNH erythrocytes of types I, II and III using flow cytometry;
· biochemical blood test (total bilirubin, direct bilirubin, LDH);
· Coombs test;
· myelogram.

Additional diagnostic examinations carried out on an outpatient basis:



· determination of the concentration of folic acid and vitamin B12;
· coagulogram;
standard cytogenetic examination of bone marrow;
· general urine analysis
· ELISA for markers of viral hepatitis;
· ELISA for HIV markers;
· ELISA for markers of herpes group viruses;
· HLA - typing;
· ECG;
· Ultrasound of the abdominal organs (liver, spleen, pancreas, gall bladder, lymph nodes, kidneys, in women - pelvis;

The minimum list of examinations that must be carried out when referring for planned hospitalization:
· general blood test (counting leukemia, platelets and reticulocytes in a smear);
· myelogram;
· blood type and Rh factor
· biochemical blood test (total protein, albumin, total bilirubin, direct bilirubin, creatinine, urea, ALaT, ASaT, GGTP, glucose, LDH, C-reactive protein, alkaline phosphatase);
· Coombs test;
· Ultrasound of the abdominal organs and spleen;
· Ultrasound of the pelvic organs - for women.

Basic (mandatory) diagnostic examinations carried out at the hospital level:

General blood test (counting leukemia, platelets and reticulocytes in a smear);
- immunophenotyping of peripheral blood to determine the percentage of PNH erythrocytes of types I, II and III using flow cytometry;
- biochemical blood test (total bilirubin, direct bilirubin, LDH);
- Coombs test
- myelogram.
- standard cytogenetic examination of the bone marrow;
- ELISA for markers of viral hepatitis;
- ELISA for HIV markers;
- ELISA for markers of herpes group viruses;
· X-ray of the chest organs.
Additional diagnostic examinations carried out at the hospital level:
· determination of haptoglobin level.
· blood type and Rh factor;
· biochemical blood test (total protein, albumin, total bilirubin, direct bilirubin, creatinine, urea, ALaT, ACaT, glucose, LDH, GGTP, C-reactive protein, alkaline phosphatase);
· iron metabolism (determining the level of serum iron, total iron-binding capacity of serum and ferritin level);
· Determination of the concentration of folic acid and vitamin B12;
· coagulogram;
· HLA - typing;
· general urine analysis;
· determination of hemosiderin level in urine;
· Reberg-Tareev test (determination of glomerular filtration rate);
· ECG;
· Ultrasound of the abdominal organs (liver, spleen, pancreas, gall bladder, lymph nodes, kidneys, in women - pelvis;
X-ray of the chest organs;
· Doppler ultrasound of arteries and veins;
· echocardiography;
· FGDS (dilatation of the veins of the esophagus);
daily blood pressure monitoring;
· 24-hour ECG monitoring.

Diagnostic measures carried out at the stage of emergency medical care:
· collection of complaints and medical history;
· physical examination.

Diagnostic criteria for diagnosis:

Complaints and anamnesis:
- weakness;
- fast fatiguability;


- increased bleeding.

Anamnesis: you should pay attention to:
- long-term weakness;
- rapid fatigue;
- frequent infectious diseases;
- acute attacks of pain in the lumbar region;
- darkening of urine, mainly at night and in the morning;
- Budd-Chiari syndrome (hepatic vein thrombosis);
- thrombosis of various localizations;
- increased bleeding;
- the appearance of hemorrhagic rashes on the skin and mucous membranes;
- dispensary registration for AA or MDS.

Physical examination[ 8 ]:
- a combination of pallor and yellowness of the skin;
- hemorrhagic rashes - petechiae, ecchymoses of various localizations;
- shortness of breath;
- tachycardia;
- enlarged liver;
- enlarged spleen.

Laboratory research:
If PNH is suspected, flow cytometry allows an accurate diagnosis to be made. Flow cytometry is the most sensitive and informative method.
· General blood analysis: The reticulocyte count is usually elevated, and peripheral blood smears show red blood cells morphologically no different from normal. Due to hemolysis, normoblasts are often present in the blood, and polychromatophilia is noted. As a result of significant losses of iron in the urine, patients with PNH are highly likely to develop iron deficiency, and then the red blood cells take on the appearance characteristic of IDA - hypochromic with a tendency to microcytosis. The number of leukocytes and platelets is often reduced. Pancytopenia of varying severity may also be observed. However, unlike aplastic anemia, reticulocytosis usually occurs along with cytopenia.
· Blood chemistry: The amount of bilirubin, free hemoglobin and methemoglobin in the blood serum is increased. There are signs of intravascular hemolysis, that is, a decrease or absence of haptoglobin, an increase in LDH, an increased level of free hemoglobin and iron in the urine. Low haptoglobin levels are consistently observed in intravascular hemolysis but also occur in cases of extravascular hemolysis, especially chronic hemolysis. Since haptoglobin is also an acute-phase reagent, its sharp decrease or absence is most informative.
· In urine: Hematuria and proteinuria may be detected. Constant signs of diagnostic significance are hemosiderinuria and the detection of blood detritus in the urine.
· Morphological study: Erythroid hyperplasia is detected in the bone marrow. Bone marrow hypoplasia and a reduced content of siderocytes and sideroblasts are often detected.
· Immunophenotyping: An early and reliable sign of the PNH phenotype is the expression of GPI-related proteins: the expression of CD14 and CD48 is determined on monocytes, CD16 and CD66b - on granulocytes, CD48 and CD52 - on lymphocytes, CD55 and CD59 - on erythrocytes, CD55, CD58.

Instrumental studies:
· Ultrasound of the abdominal organs: increase in the size of the liver and spleen.
· Doppler ultrasound of arteries and veins: the presence of thrombosis of arteries and veins
· ECG: Impaired conduction of impulses in the heart muscle.
· EchoCG: signs of heart failure (HF)<60%), снижение сократимости, диастолическая дисфункция, легочная гипертензия, пороки и регургитации клапанов.
· Whole body CT/MRI: detection of thrombosis (cerebral, portal, etc.)
· CT scan of the thoracic segment: infiltrative changes in lung tissue, signs of pulmonary hypertension.
· FGDS: varicose veins of the esophagus.
· Spirography: Pulmonary function testing.

Indications for consultation with specialists:
· doctor for x-ray endovascular diagnostics and treatment - installation of a central venous catheter from a peripheral access (PICC);
· hepatologist - for the diagnosis and treatment of viral hepatitis;
· gynecologist - pregnancy, metrorrhagia, menorrhagia, consultation when prescribing combined oral contraceptives;
· dermatovenerologist - skin syndrome No.
· infectious disease specialist - suspicion of viral infections;
· cardiologist - uncontrolled hypertension, chronic heart failure, heart rhythm and conduction disorders;
· neurologist acute cerebrovascular accident, meningitis, encephalitis, neuroleukemia;
· neurosurgeon - acute cerebrovascular accident, dislocation syndrome;
· nephrologist (efferentologist) - renal failure;
· oncologist - suspicion of solid tumors;
otorhinolaryngologist - for the diagnosis and treatment of inflammatory diseases of the paranasal sinuses and middle ear;
· ophthalmologist - visual impairment, inflammatory diseases of the eye and appendages;
· proctologist - anal fissure, paraproctitis;
· psychiatrist - psychosis;
· psychologist - depression, anorexia, etc.;
· resuscitator - treatment of severe sepsis, septic shock, acute pulmonary injury syndrome with differentiation syndrome and terminal conditions, installation of central venous catheters.
· rheumatologist - Sweet's syndrome;
· thoracic surgeon - exudative pleurisy, pneumothorax, pulmonary zygomycosis;
· transfusiologist - for the selection of transfusion media in case of a positive indirect antiglobulin test, ineffective transfusions, acute massive blood loss;
· urologist - infectious and inflammatory diseases of the urinary system;
· phthisiatrician - suspicion of tuberculosis;
· surgeon - surgical complications (infectious, hemorrhagic);
· maxillofacial surgeon - infectious and inflammatory diseases of the dentofacial system.

Differential diagnosis

Differential diagnosis.
Differential diagnosis is made with other types of hemolytic anemia, and with the cytopenic variant of PNH - with aplastic anemia.

B-12 deficiency anemia. Often there is a need for differential diagnosis of PNH, which occurs with pancytopenia and hemolysis, and B12-deficiency anemia with hemolytic syndrome. In both of these diseases, hemolysis is quite pronounced. The differences between these diseases are presented in the table:

Table. Differential diagnostic differences between B12-deficiency anemia and PNH.

Treatment

Treatment goals:
Achieving and maintaining remission (see paragraph 15 - Indicators of treatment effectiveness).

Treatment tactics:
Non-drug treatment:
Mode II: general security.
Diet: Neutropenic patients are not recommended to follow a specific diet ( level of evidence B).

Drug treatment.
The general algorithm for the treatment of patients with PNH, depending on the form of the disease and the severity of hemolysis, is presented in the figure.

Treatment algorithm for patients with PNH.


Eclizumab therapy.
Eculizumab is a humanized monoclonal antibody that binds to the C5 component of complement. This prevents the cleavage of C5 into C5a and C5b, thereby inhibiting the formation of pro-inflammatory cytokines (via C5a) and MAC (via C5b).
Currently, one multicenter, randomized, double-blind, placebo-controlled trial, TRIUMPH, assessed the effectiveness of eculizumab in stabilizing hemoglobin levels and reducing transfusion dependence in 87 transfusion-dependent patients with PNH over 6 months of therapy.
The study included patients over 18 years of age who had undergone at least 4 transfusions of erythrocyte-containing media over the past year, with an erythrocyte PNH clone type III of at least 10%, a platelet level of at least 100 thousand/μl, and an increase in LDH of ³1.5 normal. All patients received an anti-meningococcal vaccine before starting therapy.
The main result of the study was stabilization of hemoglobin levels in 49% of patients receiving eculizumab (p<0,001) и снижение необходимости в трансфузиях в этой группе до нуля (в группе плацебо за 6 месяцев потребовалось от 6 до 16 трансфузий), а также улучшение качества жизни.
The results of this study provided the basis for FDA approval of eculizumab for transfusion-dependent PNH with hemolysis.
A study by R. Hillman et al. and subsequent prospective studies have certain limitations that make it difficult to extrapolate their results to all patients with PNH, which are described in detail in the FDA report and Cochrane review by Arturo J Martí-Carvajal:
· Efficacy was studied only in patients over 18 years of age;
· Data on elderly patients is also limited (only 15 patients in the study were over 65 years old);
· The study included only transfusion-dependent patients with hemolysis;
· The small number of patients with thrombotic episodes and the high frequency of prescription of anticoagulant prophylaxis do not allow us to assess the effect of eculizumab on the risk of thrombotic complications and recommend against the use of anticoagulants in patients receiving eculizumab. The relative reduction in the frequency of thrombotic episodes during anticoagulant prophylaxis and eculizumab therapy is 81%;
· The quality of life questionnaire used has not been validated for patients with PNH and the improvement in quality of life could only be associated with an increase in hemoglobin levels;
· Short observation period;
· The study was sponsored by the manufacturer of the drug;
· There are no data on the effect of eculizumab compared with placebo on overall survival, the risk of transformation to AML and MDS. An increase in overall survival was shown in only one study with historical control (period from 1997 to 2004). In 2013, data from three prospective studies of 195 patients with PNH and hemolysis were published and showed a 97.6% survival rate at 36 months, but no comparison with a placebo group was made.
· Data on the use of eculizumab in pregnant women are limited. Pregnancy increases the incidence of severe life-threatening complications of PNH. There is a high probability that eculizumab crosses the blood-placental barrier and breast milk. Due to the rarity of the disease, there are currently no controlled studies of the effectiveness of eculizumab in pregnant women. Two cases of prescribing eculizumab to pregnant women from 4 and 5 weeks of gestation with subsequent uncomplicated pregnancies and the birth of healthy children are described.
· Even with long-term treatment, lasting about 30 months, about 18% of patients remain transfusion dependent. A possible explanation for this phenomenon is the participation of the C3 fragment of complement in the processes of intravascular hemolysis, which is not inhibited by eculizumab.

Eculizumab may be recommended for inclusion in the treatment program for the following categories of patients with classic PNH over 18 years of age:
Transfusion dependence due to chronic hemolysis ( level of evidence A);
presence of thrombotic complications ( level of evidenceD);
pregnancy in patients with PNH ( level of evidenceD).

When determining indications for eculizumab therapy, LDH levels alone should not be taken into account.

Method of administration and dosage of eculizumab
The drug is administered intravenously, by drip, over 25-45 minutes - for adults.
The course of treatment includes a 4-week initial cycle followed by a maintenance cycle. The initial cycle is 600 mg of the drug once a week for 4 weeks. Maintenance therapy - 900 mg in the 5th week, followed by 900 mg of the drug every (14±2) days.

"Breakthrough" hemolysis.
The standard eculizumab therapy regimen is sufficient for complete and stable blockade of complement-mediated hemolysis. In some patients, due to
peculiarities of drug metabolism or during infections, “breakthrough” hemolysis may develop. In this situation, signs of hemolysis appear within 2-3 days
before the next administration of eculizumab. Patients may develop hemoglobinuria, return of the original symptoms (shortness of breath, weakness, spasm of smooth muscles, etc.), the need for transfusions, increase in the level of LDH, reticulocytes and decrease in the level of haptoglobin. Treatment of breakthrough hemolysis involves reducing the interval between administrations of eculizumab to 12 days or increasing the dose to 1200 mg for 1-2 administrations.

Prevention and treatment of menincococcal infection.
During treatment with eculizumab, it is necessary to monitor the appearance of symptoms of infection and promptly prescribe antibiotics for bacterial infections. If meningococcal infection is diagnosed, the next administration of the drug is canceled.
The mechanism of action of the drug eculizumab suggests an increased risk of developing meningococcal infection ( Neisseria meningitidis) against the background of its use (level of evidence B).
All patients must be vaccinated against meningococcus 2 weeks before starting the drug, as well as revaccination between 2.5-3 years of therapy. The most preferred tetravalent conjugate vaccine is against serotypes A, C, Y and W135. If urgent treatment with eculizumab is necessary in an unvaccinated patient, therapy may be initiated against the background of appropriate antibiotic prophylaxis, which should continue for 2 weeks after vaccination against meningococcal infection.

Symptomatic therapy.
When treating with eculizumab, symptomatic therapy includes the administration of folic acid (5 mg/day), vitamin B12 (for deficiency), iron supplements (for deficiency), anticoagulants (warfarin, low molecular weight heparin) for thrombotic complications, transfusions of blood products depending on clinical symptoms, hydration during the development of a hemolytic crisis. Iron supplements should be prescribed with caution due to the possibility of increased hemolysis.

Anticoagulant therapy.
After a thrombotic event, long-term (lifelong) therapy with anticoagulants (coumarin derivatives or heparins) may be recommended. Therapy for Budd-Chiari syndrome requires the patient to be in a specialized surgical department for local and systemic thrombolysis. Anticoagulant therapy for primary prevention of thrombosis may be indicated in selected cases when a PNH clone is detected in ≥ 50% of granulocytes and in the presence of additional risks of thrombotic complications, with the exception of patients with bone marrow aplasia.

Transfusion support.
Indications for transfusion of blood components:

Erythrocyte suspension/mass.
· in relation to erythrocyte suspension/mass, selection by blood type and Rh factor is necessary;
· for patients with a history of multiple transfusions, it is advisable to select for the following antigens: Kell, Duffy, Kidd, MNSs;
· immediately before transfusion of erythrocyte suspension/mass, it is necessary to conduct a compatibility test with standard sera;
· threshold values ​​at which the need for transfusion of red blood cell suspension/mass is considered: Hb<80 г/мл, Ht <25%;
· calculation of the maximum volume of erythrocyte suspension/mass is determined by the following formula: Hb (g/dL) x4 x recipient weight (kg).

Platelet concentrate.
· Platelet concentrate must be selected according to blood type and Rh factor;
· transfusion of platelet concentrate to prevent bleeding, carried out at Tr level<10 тыс кл/мкл;
· patients with febrile fever, bleeding of mucous membranes are recommended to undergo transfusion of platelet concentrate at Tr level<20 тыс кл/мкл;
· when planning an invasive intervention for a patient, it is recommended to carry out a transfusion of platelet concentrate at the Tr level<50 тыс кл/мкл;
· therapeutic dose of platelets recommended for adults: 3 x 10 11 cells/l in a volume of 200-300 ml.

Assessing the effectiveness of transfusion:
stopping bleeding;
Determination of platelet level the next day - persistent Tr level<20 тыс кл/мкл свидетельствует о рефрактерности к трансфузиям;
· if all causes of thrombocytopenia are excluded, it is necessary to test for the presence of anti-leukocyte antibodies;
· If antibodies are detected, platelet transfusion must be carried out from an HLA-compatible donor.

Fresh frozen plasma.
Since FFP contains complement, its transfusions can provoke the development of hemolysis in patients with PNH. It is advisable to avoid transfusions of FFP in PNH.

Drug treatment provided on an outpatient basis:
− list of essential medicines indicating the release form (having a 100% probability of use):

Antineoplastic and immunosuppressive drugs
. eculizumab*300 mg, concentrate for solution for infusion, 10 mg/ml.

· filgrastim, solution for injection 0.3 mg/ml, 1 ml;
· ondansetron, solution for injection 8 mg/4ml.

Antibacterial agents
Azithromycin, tablet/capsule, 500 mg;
· amoxicillin/clavulanic acid, film-coated tablet, 1000 mg;
· moxifloxacin, tablet, 400 mg;
Ofloxacin, tablet, 400 mg;
· ciprofloxacin tablet, 500 mg;
· metronidazole, tablet, 250 mg, dental gel 20g;
· erythromycin, tablet 250 mg.

· anidulafungin, lyophilized powder for solution for injection, 100 mg/vial;



· clotrimazole, solution for external use 1% 15ml;

Fluconazole, capsule/tablet 150 mg.

· acyclovir, tablet, 400 mg, gel in tube 100,000 units 50 g;


Famciclovir, tablets, 500 mg.

Solutions used to correct disturbances in water, electrolyte and acid-base balance

· dextrose, solution for infusion 5% 250ml;
· sodium chloride, solution for infusion 0.9% 500ml.

· heparin, solution for injection 5000 IU/ml, 5 ml; (for flushing the catheter)

· rivaroxaban, tablet;
tranexamic acid, capsule/tablet 250 mg;

· ambroxol, solution for oral administration and inhalation, 15 mg/2 ml, 100 ml;

· atenolol, tablet 25 mg;



· drotaverine, tablet 40 mg;


Levofloxacin, tablet, 500 mg;

Lisinopril, 5 mg tablet;
· methylprednisolone, tablet, 16 mg;

· omeprazole, capsule 20 mg;

Prednisolone, tablet, 5 mg;
· dioctahedral smectite, powder for preparation of suspension for oral administration 3.0 g;

· torasemide, tablet 10 mg;
· fentanyl, therapeutic transdermal system 75 mcg/h; (for the treatment of chronic pain in cancer patients)

Drug treatment provided at the inpatient level:
− list of essential medicines indicating the release form (having a 100% probability of use):

· eculizumab*300 mg, concentrate for solution for infusion, 10 mg/ml.

− list of additional medicines indicating the release form (less than 100% probability of use):

Medicines that weaken the toxic effect of anticancer drugs
. filgrastim, solution for injection 0.3 mg/ml, 1 ml;
. ondansetron, solution for injection 8 mg/4ml.

Antibacterial agents
· azithromycin, tablet/capsule, 500 mg, lyophilized powder for the preparation of solution for intravenous infusion, 500 mg;
· amikacin, powder for injection, 500 mg/2 ml or powder for solution for injection, 0.5 g;
· amoxicillin/clavulanic acid, film-coated tablet, 1000 mg, powder for solution for intravenous and intramuscular administration 1000 mg+500 mg;
· vancomycin, powder/lyophilisate for solution for infusion 1000 mg;
· gentamicin, solution for injection 80 mg/2 ml 2 ml;
· imipinem, cilastatin powder for solution for infusion, 500 mg/500 mg;
· sodium colistimethate*, lyophilisate for the preparation of solution for infusion, 1 million units/bottle;
· metronidazole tablet, 250 mg, solution for infusion 0.5% 100 ml, dental gel 20 g;
Levofloxacin, solution for infusion 500 mg/100 ml, tablet 500 mg;
linezolid, solution for infusion 2 mg/ml;
· meropenem, lyophilisate/powder for solution for injection 1.0 g;
· moxifloxacin, tablet 400 mg, solution for infusion 400 mg/250 ml
· ofloxacin, tablet 400 mg, solution for infusion 200 mg/100 ml;
· piperacillin, tazobactam powder for solution for injection 4.5 g;
tigecycline*, lyophilized powder for solution for injection 50 mg/bottle;
Ticarcillin/clavulanic acid, lyophilized powder for the preparation of solution for infusion 3000 mg/200 mg;
cefepime, powder for solution for injection 500 mg, 1000 mg;
· cefoperazone, sulbactam powder for solution for injection 2 g;
· ciprofloxacin, solution for infusion 200 mg/100 ml, 100 ml, 500 mg tablet;
· erythromycin, tablet 250 mg;
Ertapenem lyophilisate, for the preparation of a solution for intravenous and intramuscular injections 1 g.

Antifungal drugs
· amphotericin B*, lyophilized powder for solution for injection, 50 mg/vial;
· anidulofungin, lyophilized powder for solution for injection, 100 mg/vial;
voriconazole, powder for solution for infusion 200 mg/bottle;
voriconazole, tablet, 50 mg;
· itraconazole, oral solution 10 mg/ml 150.0;
· caspofungin, lyophilisate for the preparation of solution for infusion 50 mg;
· clotrimazole, cream for external use 1% 30g, solution for external use 1% 15ml;
· micafungin, lyophilized powder for the preparation of solution for injection 50 mg, 100 mg;
· fluconazole, capsule/tablet 150 mg, solution for infusion 200 mg/100 ml, 100 ml.

Antiviral drugs
· acyclovir, cream for external use, 5% - 5.0, tablet - 400 mg, powder for solution for infusion, 250 mg;
· valacyclovir, tablet, 500 mg;
· valganciclovir, tablet, 450 mg;
· ganciclovir*, lyophilisate for solution for infusion 500 mg;
Famciclovir, tablets, 500 mg No. 14.

Medicines used for pneumocystosis
· sulfamethoxazole/trimethoprim, concentrate for solution for infusion (80mg+16mg)/ml, 5 ml;
· sulfamethoxazole/trimethoprim, tablet 480 mg.

Additional immunosuppressive drugs:
· dexamethasone, solution for injection 4 mg/ml 1 ml;
· methylprednisolone, tablet 16 mg, solution for injection 250 mg;
· prednisolone, solution for injection 30 mg/ml 1 ml, tablet 5 mg.

Solutions used to correct disturbances of water, electrolyte and acid-base balance, parenteral nutrition
· albumin, solution for infusion 10%, 100 ml;
· albumin, solution for infusion 20% 100 ml;
· water for injection, solution for injection 5 ml;
· dextrose, solution for infusion 5% - 250 m, 5% - 500 ml; 40% - 10 ml, 40% - 20 ml;
· potassium chloride, solution for intravenous administration 40 mg/ml, 10 ml;
· calcium gluconate, solution for injection 10%, 5 ml;
· calcium chloride, solution for injection 10% 5ml;
· magnesium sulfate, solution for injection 25% 5 ml;
· mannitol, solution for injection 15% -200.0;
· sodium chloride, solution for infusion 0.9% 500ml;
· sodium chloride, solution for infusion 0.9% 250ml;
· sodium chloride, potassium chloride, sodium acetate solution for infusion in a bottle of 200 ml, 400 ml;
· sodium chloride, potassium chloride, sodium acetate solution for infusion 200ml, 400ml;
· sodium chloride, potassium chloride, sodium bicarbonate solution for infusion 400ml;
L-alanine, L-arginine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L- tryptophan, L-tyrosine, L-valine, sodium acetate trihydrate, sodium glycerophosphate pentihydrate, potassium chloride, magnesium chloride hexahydrate, glucose, calcium chloride dihydrate, olive and soybean oils emulsion mixture for inf.: three-chamber containers 2 l
· hydroxyethyl starch (pentastarch), solution for infusion 6% 500 ml;
· amino acid complex, emulsion for infusion containing a mixture of olive and soybean oils in a ratio of 80:20, a solution of amino acids with electrolytes, a dextrose solution, with a total calorie content of 1800 kcal 1,500 ml three-section container.

Medicines used for intensive therapy (cardiotonic drugs for the treatment of septic shock, muscle relaxants, vasopressors and anesthetics):
· aminophylline, solution for injection 2.4%, 5 ml;
· amiodarone, solution for injection, 150 mg/3 ml;
· atenolol, tablet 25 mg;
· atracurium besylate, solution for injection, 25 mg/2.5 ml;
· atropine, solution for injection, 1 mg/ml;
· diazepam, solution for intramuscular and intravenous use 5 mg/ml 2 ml;
· dobutamine*, solution for injection 250 mg/50.0 ml;
· dopamine, solution/concentrate for the preparation of solution for injection 4%, 5 ml;
· simple insulin;
· ketamine, solution for injection 500 mg/10 ml;
· morphine, solution for injection 1% 1 ml;
· norepinephrine*, solution for injection 20 mg/ml 4.0;
· pipecuronium bromide, lyophilized powder for injection 4 mg;
· propofol, emulsion for intravenous administration 10 mg/ml 20 ml, 10 mg/ml 50 ml;
· rocuronium bromide, solution for intravenous administration 10 mg/ml, 5 ml;
· sodium thiopental, powder for the preparation of solution for intravenous administration 500 mg;
· phenylephrine, solution for injection 1% 1ml;
· phenobarbital, tablet 100 mg;
human normal immunoglobulin, solution for infusion;
· epinephrine, solution for injection 0.18% 1 ml.

Medicines affecting the blood coagulation system
· aminocaproic acid, solution 5% -100 ml;
· anti-inhibitory coagulant complex, lyophilized powder for the preparation of injection solution, 500 IU;
· heparin, solution for injection 5000 IU/ml, 5 ml, gel in tube 100000 IU 50g;
· hemostatic sponge, size 7*5*1, 8*3;
· nadroparin, solution for injection in pre-filled syringes, 2850 IU anti-Xa/0.3 ml, 5700 IU anti-Xa/0.6 ml;
· enoxaparin, solution for injection in syringes 4000 anti-Xa IU/0.4 ml, 8000 anti-Xa IU/0.8 ml.

Other medicines
· bupivacaine, solution for injection 5 mg/ml, 4 ml;
· lidocaine, solution for injection, 2%, 2 ml;
· procaine, solution for injection 0.5%, 10 ml;
· human immunoglobulin normal solution for intravenous administration 50 mg/ml - 50 ml;
· omeprazole, capsule 20 mg, lyophilized powder for the preparation of solution for injection 40 mg;
· famotidine, lyophilized powder for the preparation of solution for injection 20 mg;
Ambroxol, solution for injection, 15 mg/2 ml, solution for oral administration and inhalation, 15 mg/2 ml, 100 ml;
· amlodipine, tablet/capsule 5 mg;
· acetylcysteine, powder for solution for oral administration, 3 g;
· dexamethasone, eye drops 0.1% 8 ml;
Diphenhydramine, solution for injection 1% 1 ml;
· drotaverine, solution for injection 2%, 2 ml;
· captopril, tablet 50 mg;
· ketoprofen, solution for injection 100 mg/2ml;
lactulose, syrup 667 g/l, 500 ml;
· chloramphenicol, sulfadimethoxin, methyluracil, trimecaine ointment for external use 40g;
Lisinopril, 5 mg tablet;
· methyluracil, ointment for topical use in a tube 10% 25g;
· naphazoline, nasal drops 0.1% 10ml;
· nicergoline, lyophilisate for the preparation of injection solution 4 mg;
· povidone-iodine, solution for external use 1 l;
· salbutamol, solution for nebulizer 5 mg/ml-20 ml;
· smectitedioctahedral, powder for the preparation of suspension for oral administration 3.0 g;
· spironolactone, capsule 100 mg;
· tobramycin, eye drops 0.3% 5ml;
· torasemide, tablet 10 mg;
· tramadol, solution for injection 100 mg/2ml;
tramadol, oral solution (drops) 100 mg/1 ml 10 ml;
· fentanyl, therapeutic transdermal system 75 mcg/h (for the treatment of chronic pain in cancer patients);
· folic acid, tablet, 5 mg;
· furosemide, solution for injection 1% 2 ml;
· chloramphenicol, sulfadimethoxine, methyluracil, trimecaine ointment for external use 40g;
· chlorhexidine, solution 0.05% 100ml;
· chloropyramine, solution for injection 20 mg/ml 1 ml.

Drug treatment provided at the emergency stage: is not carried out.

Other types of treatment:
Other types of treatment provided on an outpatient basis: do not apply.

Other types of services provided at the stationary level:

Bone marrow transplantation (level of evidence B)
Indications for BMT in PNH are similar to severe aplastic anemia.
While eculizumab helps control intravascular hemolysis and associated complications of PNH, primarily transfusion dependence, allogeneic bone marrow transplantation (BMT) remains the only radical method to achieve a cure for this disease. However, BMT is associated with high mortality. Thus, in a retrospective study on 26 patients with PNH from Italy who received BMT, the 10-year survival rate was 42%, and the probability of 2-year survival in 48 patients who received BMT from an HLA-identical sibling, according to the International Bone Marrow Transplantation Registry, amounted to 56%. Regardless of the indications for which BMT is performed, the incidence of complications remains very high. The incidence of graft-versus-host disease in patients with PNH is 42-54%, half of the patients develop veno-occlusive liver disease, non-engraftment or rejection and, in addition, the risk of expansion of the PNH clone remains. BMT and associated complications negatively affect the quality of life of patients.

Other types of treatment provided during emergency medical care: do not apply.

Features of management of pregnant patients.
Pregnancy in PNH is associated with high levels of maternal and child mortality (11.6% and 7.2%, respectively).
Currently, only isolated cases of eculizumab therapy during pregnancy with a favorable outcome for the mother and fetus have been described. There are no teratogenic effects of the drug. During pregnancy, eculizumab therapy should not be discontinued. If the patient has not previously received eculizumab, the drug can be prescribed during pregnancy. In this case, eculizumab therapy should be continued for 3 months after delivery. In cases of breakthrough hemolysis during pregnancy, a dose adjustment of the drug may be required (for example, maintenance therapy 900 mg per week).

Surgical intervention:
Surgical intervention provided on an outpatient basis: is not carried out.

Surgical intervention provided in an inpatient setting:
With the development of infectious complications and life-threatening bleeding, patients undergo surgical interventions for emergency indications.

Further management:
During therapy with eculizumab, the following laboratory tests are recommended: complete blood count with determination of reticulocytes, LDH, blood creatinine, brain natriuretic peptide B (if possible), D-dimer, serum iron, ferritin, direct antiglobulin test. The size of the PNH clone is monitored based on the results of highly sensitive flow cytometry.
In patients receiving eculizumab, a statistically significant increase in the size of the PNH clone is observed. In the TRIUMPH study, the type III PNH clone of erythrocytes increased from 28.1% to 56.9% over 26 weeks, while it did not change in the placebo group. If eculizumab is discontinued, monitoring of the size of the PNH clone, the level of reticulocytes, haptoglobin, LDH, bilirubin, and D-dimers is necessary for timely detection of hemolysis and prevention of potential complications.

Indicators of treatment effectiveness:
A specific system for assessing the response to therapy in PNH has not yet been developed. When assessing the effect of treatment, the following are taken into account:
· clinical manifestations - weakness;
· hemoglobin level;
· the need for transfusions of blood components;
thrombotic episodes;
· hemolysis activity (level of reticulocytes, LDH, haptoglobin).

Drugs (active ingredients) used in treatment

Groups of drugs according to ATC used in treatment

Hospitalization

Indications for hospitalization:
Indications for emergency hospitalization:
· newly diagnosed PNH;
· thrombotic complications;
· hemolytic crisis;
febrile neutropenia.

Indications for planned hospitalization:
· examination, determination of further treatment tactics;
allogeneic bone marrow transplantation.

Prevention

Preventive actions: No.

Information

Sources and literature

1. Minutes of meetings of the Expert Council of the RCHR of the Ministry of Health of the Republic of Kazakhstan, 2015
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Information

List of protocol developers with qualification details:

1) Kemaikin Vadim Matveevich - Candidate of Medical Sciences, JSC "National Scientific Center of Oncology and Transplantology", Head of the Department of Oncohematology and Bone Marrow Transplantation.
2) Anton Anatolyevich Klodzinsky - Candidate of Medical Sciences, JSC National Scientific Center of Oncology and Transplantology, hematologist at the Department of Oncohematology and Bone Marrow Transplantation.
3) Ramazanova Raigul Mukhambetovna - Doctor of Medical Sciences, Professor of JSC "Kazakh Medical University of Continuing Education", head of the hematology course.
4) Gabbasova Saule Telembaevna - RSE at the RSE "Kazakh Research Institute of Oncology and Radiology", head of the department of hemoblastosis.
5) Karakulov Roman Karakulovich - Doctor of Medical Sciences, Professor, Academician of the MAI RSE at the Kazakh Research Institute of Oncology and Radiology, chief researcher of the department of hemoblastosis.
6) Tabarov Adlet Berikbolovich - Head of the Department of Innovative Management of the RSE at the RSE "Hospital of the Medical Center Administration of the President of the Republic of Kazakhstan", clinical pharmacologist, pediatrician.
7) Rapilbekova Gulmira Kurbanovna, Doctor of Medical Sciences. JSC “National Scientific Center for Maternity and Childhood” - head of the obstetric department No. 1.

Disclosure of no conflict of interest: absent.

Reviewers:
1) Afanasyev Boris Vladimirovich - Doctor of Medical Sciences, Director of the Research Institute of Children's Oncology, Hematology and Transplantology named after R.M. Gorbacheva, Head of the Department of Hematology, Transfusiology and Transplantology, State Budgetary Institution of Higher Professional Education, First St. Petersburg State Medical University named after. I.P. Pavlova.
2) Rakhimbekova Gulnar Ayapbekkyzy - Doctor of Medical Sciences, Professor, JSC National Scientific Medical Center, Head of Department.
3) Pivovarova Irina Alekseevna - Medicinae Doctor, Master of Business Administration, Chief freelance hematologist of the Ministry of Health and Social Development of the Republic of Kazakhstan.

Indication of the conditions for reviewing the protocol: revision of the protocol after 3 years and/or when new diagnostic and/or treatment methods with a higher level of evidence become available.

Attached files

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Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disease manifested by persistent hemolytic anemia, paroxysmal or persistent hemoglobinuria, and intravascular hemolysis. The rarity of this type of hemolytic anemia is characterized by the fact that PNH affects 1 person in half a million, mostly young people.

The causes of the disease are currently unknown. It is assumed that it occurs due to the occurrence of an abnormal clone of red blood cells prone to intravascular hemolysis. In turn, the inferiority of red blood cells is a consequence of structural and biochemical defects in their membrane. It is known that lipid peroxidation is activated in a defective membrane, which promotes rapid lysis of red blood cells; in addition, abnormal clones of granulocytes and platelets are involved in the pathological process. The main role in the occurrence of thrombotic complications of PNH belongs to the intravascular destruction of erythrocytes and the initiation of blood coagulation by factors released during this process. PNH, as a rule, begins gradually and proceeds chronically with periodic crises. Crises are provoked by viral infections, surgical interventions, psycho-emotional stress, menstruation, and the use of a number of medications and foods.

Symptoms of paroxysmal nocturnal hemoglobinuria

Symptoms of PNH during a crisis:

• paroxysmal pain in the abdominal cavity;
• pain in the lumbar region;
• icterus of the skin and sclera; hyperthermia; facial pastiness;
• black color of urine, mainly at night;
• a sharp decrease in blood pressure;
• transient enlargement of the spleen;
• cessation of urine output.

In some cases, the hemolytic crisis ends in death.

Symptoms of PNH outside of crisis:

• general weakness;
• pale skin color with a jaundiced tint;
• anemia;
• tendency to thrombosis; hematuria; high blood pressure; liver enlargement; dyspnea; heartbeat; frequent infectious diseases.

Diagnostics

• Blood test: anemia (normochromic, later hypochromic), moderate leukocytopenia and thrombocytopenia, serum iron level is significantly reduced.
• Examination of urine: black staining, hemoglobinuria, hemosiderinuria, proteinuria. The Gregersen urine benzidine test is positive.
• Ham's specific test is positive.
• The specific Hartmann test is positive.
• Bone marrow puncture: hyperplasia of the red hematopoietic lineage, but in severe cases, bone marrow hypoplasia and an increase in the amount of adipose tissue in the bone marrow can also be observed.

Treatment of paroxysmal nocturnal hemoglobinuria

Treatment of PNH is symptomatic and consists mainly of replacement blood transfusions, the volume and frequency of which depend on the “response” to these measures. In the treatment of PNH, methandrostenolone is used at a dose of 30-50 mg/day for at least 2-3 months. The fight against bone marrow hypoplasia is carried out by intravenous use of antithymocyte immunoglobulin at a dose of 150 mg/day for 4 to 10 days. It is recommended to take iron supplements per os in small dosages. Sometimes corticosteroids in high dosages have a good effect. Bone marrow hypoplasia with the development of thrombotic complications are indications for its transplantation. Isolated cases of recovery from PNH have been described; in some cases, the duration of a favorable course of the disease is several decades.

Essential drugs

There are contraindications. Specialist consultation is required.