PNG clone analysis. Method for laboratory diagnosis of paroxysmal nocturnal hemoglobinuria. Video - Paroxysmal nocturnal hemoglobinuria

1 KGBUZ "Krasnoyarsk Interdistrict Clinical Hospital No. 7"

2 Krasnoyarsk State Medical University named after Professor V.F. Voyno-Yasenetsky" of the Ministry of Health of the Russian Federation

The article presents a case of successful treatment of a patient with a super-severe form of aplastic anemia in combination with paroxysmal nocturnal hemoglobinuria and chronic hemolysis. The interest of the clinical case is that the patient was simultaneously treated for severe intravascular hemolysis caused by paroxysmal nocturnal hemoglobinuria and a severe form of aplastic anemia. Given the severity of intravascular hemolysis before and after the procedure of allogeneic bone marrow transplantation, the patient underwent a short induction course of therapy with Soliris (Eculizumab) 600 mg/day into the vein D-8, D-1, D+10. Allogeneic bone marrow transplantation of the patient was performed from a sibling (sister). Currently, the functioning of the graft is satisfactory, without the need for growth factors and replacement blood transfusions. No manifestations of graft-versus-host disease were observed, and immunosuppressive therapy with cyclosporine A was continued.

aplastic anemia

paroxysmal nocturnal hemoglobinuria

bone marrow transplant (tcm)

blood diseases

2. Kulagin A.D. Aplastic anemia: immunopathogenesis, clinic, diagnosis, treatment / A.D. Kulagin, I.A. Lisukov, V.A. Kozlov. - Novosibirsk: Nauka, 2008. - 236 p.

3. Development of aplastic anemia: why and what to do? [Electronic resource]. – Access mode: http://asosudy.ru/anemiya/razvitie-aplasticheskoj-anemii (date of access: 06/28/2017).

4. Kulagin A.D., Lisukov I.A., Ptushkin V.V. et al. National Clinical Guidelines for the Diagnosis and Treatment of Paroxysmal Nocturnal Hemoglobinuria / A.D. Kulagin, I.A. Lisukov, V.V. Ptushkin // Oncohematology. - 2014. - No. 2. - P. 3-11.

5. Paroxysmal nocturnal hemoglobinuria: Information and review brochure for hematologists / ed. Kulagina A.D. – Scientific Society for Medical Innovations. – Moscow: Literature, 2015. – 29 p.

6. Kelly R. The pathophysiology of paroxysmal nocturnal hemoglobinuria and treatment with eculizumab / R. Kelly, S. Richards, P. Hillmen, A. Hill // Ther. Clin. risk management. – 2009. – V.2009:5. – P. 911-921.

7. Parker C.J. Bone marrow failure syndromes: paroxysmal nocturnal hemoglobinuria / C.J. Parker // Hematol. oncol. Clin. North Am. – 2009; 23:333-46.

8. Hillmen P. Long-term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal hemoglobinuria / P. Hillmen, P. Muus, A. Roth et al. // Br. J. Haemotol. – 2013. –162(1). – P. 62-73.

This disease was first described in 1888 by Paul Ehrlich. The name aplastic anemia was proposed in 1904 by Chauford. The frequency of the annual occurrence of the disease is from 6 to 13 cases per 1,000,000 population. Aplastic anemia (AA) is a blood disease in which pancytopenia is formed as a result of inhibition of bone marrow hematopoiesis. Immune aggression directed at hematopoietic progenitor cells due to the activity of T-lymphocytes and killers is the main mechanism of hematopoietic disorders in aplastic anemia. There is an overproduction of cytokines that suppress hematopoietic cells and stimulate the activation of T-lymphocytes. In micropreparations of the bone marrow in aplastic anemia, complete devastation of the bone marrow is noted, there are small foci of hematopoiesis. The bone marrow microenvironment plays a large role in the development of hematopoietic cells and in the functioning of the bone marrow, which in turn depends on the brain microcirculation network. The density of bone marrow vessels (density of microcirculation) in patients with aplastic anemia is low. It plays a role in the pathophysiology of brain failure. It is possible that the use of pro-angiogenic agents in the treatment of aplastic anemia will play a role in restoring bone marrow function.

Treatment of aplastic anemia:

1. Therapy aimed at restoring the bone marrow.
2. Replacement therapy with blood components, treatment and prevention of infectious complications.
3. Additional treatments for aplastic anemia

• steroids;
• splenectomy;
• colony stimulating factors.

Paroxysmal nocturnal hemoglobinuria (PNH) - Harley's disease, Marchiafava - Micheli disease, Strübing - Marchiafava disease - is an acquired, progressive systemic disease in which intravascular hemolysis is observed. The incidence of PNH reaches approximately 1 case per 1,000,000 inhabitants per year. The reason is a somatic mutation in the stem cell, total cytopenia develops with the involvement of platelets and leukocytes in the process. Thrombosis is formed, the activity of many organs is disrupted, including the physiological and immune failure of the bone marrow. It should be remembered that differential diagnosis of paroxysmal nocturnal hemoglobinuria in patients with cytopenia should be carried out. Since acquired insufficiency of hematopoietic cells can be differentiated between diseases such as aplastic anemia, poroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, as well as age-related bone marrow degeneration in perfectly healthy people, and syndromes with a non-tumor process, it is necessary, if possible, to clarify the pathogenetic mutation by sequencing with the syndrome of hematopoietic insufficiency.

There are three forms of paroxysmal nocturnal hemoglobinuria:

• Classical form with signs of hemolysis.
• PNH in patients with aplastic anemia.

Subclinical form of the disease in patients without clinical and laboratory signs of hemolysis, but in the presence of a small clone of cells with signs of Paroxysmal nocturnal hemoglobinuria.

Treatment of Paroxysmal nocturnal hemoglobinuria, presented earlier: transfusion of blood components, therapy for hemolysis, iron deficiency, stimulants for the development of young forms of erythrocytes, anti-inflammatory drugs. Mainly, the therapy was symptomatic and poliative.

Allogeneic bone marrow transplantation (BMT) is currently the only radical treatment for Paroxysmal nocturnal hemoglobinuria. TCM is associated with high mortality and complications. In addition, in the post-transplant period, sometimes there is a recovery of the PNH clone and a recurrence of Paroxysmal nocturnal hemoglobinuria.

Due to the high risk of complications, allo-TKM is performed for hematopoietic aplasia (AA / PNH and AA / subclinical PNH), as well as for malignant clonal transformation of Paroxysmal nocturnal hemoglobinuria into Myelodysplastic syndrome, acute leukemia.

Eculizumab (Soliris®, AlexionPharmaceuticals, Cheshire, CT) is currently the only effective pathogenic therapy for paroxysmal nocturnal hemoglobinuria. Eculizumab is a humanized monoclonal antibody that binds to complement, which prevents the cleavage of the C-component of complement, while inhibiting the formation of the membrane attack complex. The effectiveness of the appointment has been proven only in patients who have undergone a transfusion of blood or its components. The drug is used with caution in patients who are carriers or who have an active form of meningococcal Neisseria infection with congenital complement deficiency. It is also necessary to carefully use the drug in patients with renal and hepatic insufficiency. Although there are reports of successful use of eculizumab in patients with chronic renal failure. The appointment of Soliris in patients with paroxysmal nocturnal hemoglobinuria reduces the risk of thrombosis, hemolytic complications, relieves the frequency of pulmonary hypertension, weakness, and apnea. The drug has no effect on the aplastic symptom in paroxysmal nocturnal hemoglobinuria. The drug was recommended for use in patients, including those over 65 years of age, with caution in adolescents 12-17 years of age.

Treatment consists of an induction phase (600 mg IV for 4 weeks) and a maintenance phase (900 mg IV for week 5 and every 14 days thereafter). Soliris is safe for long-term use and can significantly reduce morbidity and mortality in patients with paroxysmal nocturnal hemoglobinuria. Currently, eculizumab is used in patients with paroxysmal nocturnal hemoglobinuria in the presence of thrombotic complications, chronic hemolysis with dysfunction of organs and systems, transfusion dependence due to chronic hemolysis, pregnancy in patients with paroxysmal nocturnal hemoglobinuria.

Aplastic anemia, according to epidemiological studies, occurs in Europe, North America, the Far and Middle East. Quite often Aplastic anemia is common in Korea. In European countries, the prevalence of aplastic anemia is 2 cases per 1 million population per year, with this indicator fluctuating depending on the specific country from 0.6 to 3 or more per 1 million population per year. Aplastic anemia is often associated with paroxysmal nocturnal hemoglobinuria (PNH). A clone of Paroxysmal nocturnal hemoglobinuria in patients with aplastic anemia is found in 50%. Aplastic anemia in combination with paroxysmal nocturnal hemoglobinuria is observed in 2-4 cases per 1 million population per year.

We present a clinical case of diagnosis and successful treatment of a patient with super-severe aplastic anemia in combination with paroxysmal nocturnal hemoglobinuria.

Patient M.Ya.V., born in 1993

Diagnosis: Acquired idiopathic aplastic anemia, severe form. A course of combined immunosuppressive therapy (thymoglobulin-22.01-26.01.16) + cyclosporine A). Allogenic related bone marrow transplantation (14.06.16).

Paroxysmal nocturnal hemoglobinuria (12.2015). Chronic intravascular hemolysis.

Complications: Transient drug nephrotoxicity (CsA). Secondary hemosiderosis.

The debut of the disease since August 2015 - the appearance of heavy long menstruation up to 7 days, subcutaneous hemorrhages from minor injuries, did not seek medical help. Since November 2015, the appearance of blood in the stool.

On 12/08/2015, the patient was urgently hospitalized in the hematology department of the CMCH No. 7 in Krasnoyarsk. In the clinical picture, a pronounced anemic syndrome (pronounced general weakness, fatigue, dizziness, shortness of breath when walking and exertion); hemorrhagic syndrome (menametrorrhagia, subcutaneous hemorrhages, blood in the feces).

In the hemogram, erythrocytes 1.32 * 10 12 / l, reticulocytes 72.5% 0, spherocytosis, erythrocyte aggregation, hemoglobin 49 g / l, platelets 16 * 10 9 / l, leukocytes 2.66 * 10 9 / l, stab 1 %, segmented 4%, lymphocytes 87%, monocytes 7%, ESR 52 mm/h. Myelogram: punctate significantly reduced cellularity. Megakaryocytes were not found in the punctate. There are no blasts. Coombs test (direct, indirect) - negative. In the biochemical analysis of blood: Lactate dehydrogenase - 1194.0 U / l (N up to 450 U / l), total bilirubin 22.2 mmol / l (direct - 5.6 mmol / l, indirect 16.6 mmol / l). Trepanobiopsy: changes reflect hypoplasia of bone marrow hematopoiesis. PNH-clone was detected: among granulocytes (FLAER-CD24) - 52.72%, among monocytes (FLAER-CD14) - 58%, on erythrocytes - 11%.

Thus, on the basis of histological, cytological, enzyme-linked immunosorbent typing studies, the diagnosis was made: Acquired idiopathic aplastic anemia, super-severe form / paroxysmal nocturnal hemoglobinuria, first detected.

Conducted substitution - hemocomponent therapy (transfusion of erythrocyte suspension No. 4, thromboconcentrate No. 40), symptomatic therapy.

In December 2015, the patient was consulted in absentia by a professor of the Research Institute of Pediatric Oncology, Hematology and Transplantology (DOGiT) named after. R.M. Gorbacheva (St. Petersburg) Doctor of Medical Sciences HELL. Kulagin: HLA typing of the patient and sibling (sister) is recommended. But at that time, the sibling (sister) was 7 months pregnant. According to the results of typing, the patient and her sister are fully compatible.

In January, March 2016, repeated correspondence consultations at the Research Institute of DoGiT named after. R.M. Gorbacheva, St. Petersburg: allogeneic related bone marrow transplantation is indicated after delivery and termination of breastfeeding from a related donor. Given the severity of intravascular hemolysis, a short induction course of therapy with eculizumab is indicated to reduce the risks of allogeneic bone marrow transplantation.

Prior to allogeneic bone marrow transplantation, the patient repeatedly underwent inpatient treatment in the hematology department of the Krasnoyarsk City Clinical Hospital No. 7, where hemocomponent therapy (transfusion of erythrocyte suspension, thromboconcentrate) was performed.

During the next hospitalization in January 2016, a course of ATG (thymoglobulin 800 mg/course) + GCS was performed. The patient tolerated the therapy satisfactorily, there were no signs of serum sickness. Since March 2016, cyclosporine A has been taken at a dose of 400 mg/day, tolerability is satisfactory.

Hospitalization in the Department of Bone Marrow Transplantation, St. Petersburg State Medical University. Academician I.P. Pavlov Research Institute DogiT them. R.M. Gorbacheva - 06/06/16 Donor: sister, born in 1988, fully compatible according to the HLA system with major and minor incompatibility in ABO-AII>BIII).

Conditioning regimen: fludarabine 240 mg IV, busulfan 520 mg per os, thymoglobulin 326 mg IV. Graft-versus-host disease (GVHD) prophylaxis: cyclosporine 1560 mg IV, methotrexate 60 mg IV. Prevention of chronic intravascular hemolysis - eculizumab 600 mg/day intravenously D-8, D-1, D+10. 06/14/16 Allogeneic bone marrow transplantation was performed. The introduction of preparations of the conditioning regimen, the graft transfusion was tolerated without complications. During the early post-transplantation period, complications were observed: toxic hepatitis of the 2nd stage, against the background of the introduction of methotrexate, therapy with hepatoprotectors was carried out with positive dynamics; febrile neutropenia, response to thienam.

Restoration of peripheral blood: leukocytes >1x10 9 /l/D+24; neutrophils > 0.5x10 9 /l / D + 19, platelets > 50x10 9 / l / D + 20. Bone marrow puncture: D+28 — normocellular bone marrow, all sprouts are present, karyotype 46,XX, donor chimerism 90-97%; D+43/D+62 - all sprouts are present in the myelogram, complete donor chimerism (97%). Study of the PNH clone: ​​D+24 - a minor clone remains among granulocytes (FLAER-CD24) - 0.01%, among monocytes (FLAER-CD14) - 0.01%; there are no laboratory and clinical manifestations of intravascular hemolysis. D+43 - PNH clone is not detected among monocytes and granulocytes, among erythrocytes - minor (CD59 - 0.55%). D+65 - among monocytes and granulocytes is not detected, among erythrocytes - minor (CD59 - 0.30%).

With D+23 transferred to oral administration of cyclosporine A 200 - 250 mg/day. There were no signs of acute graft-versus-host disease. Outpatient continued taking antimicrobial, antibacterial, antifungal drugs, in order to prevent infectious complications, in the prescribed dose. The patient was observed weekly by hematologists in Krasnoyarsk, blood tests and biochemical parameters were monitored, and the dose of cyclosporine A was adjusted.

In September 2016, the patient was undergoing a routine examination and treatment at the Department of TCM for adults of the Institute of Gastrointestinal Diseases. R.M. Gorbacheva, St. Petersburg. Bone marrow puncture (D+97): normocellular bone marrow, all sprouts are present, complete donor chimerism (90-97%). Hemogram: erythrocytes 3.31*10 9 /l, hemoglobin 105 g/l, reticulocytes 1.24% 0 , platelets 165*10 9 /l, leukocytes 2.5*10 9 /l, neutrophils 71.8%. Investigation of PNH clone (D+98): PNH clone is not detected.

The functioning of the graft is satisfactory, without the need for growth factors, replacement blood transfusions. There were no manifestations of graft-versus-host disease, immunosuppressive therapy with cyclosporine A (125 mg/day) was continued, the dose was reduced due to organic toxicity (hypercreatininemia).

Repeated scheduled examination since November 2016 in the department of bone marrow transplantation for adults, IDGIT named after. R.M. Gorbacheva, St. Petersburg. Puncture of the bone marrow (D+153): moderate cellularity of the bone marrow, all sprouts are present. Hemogram: erythrocytes 3.58*10 9 /l, hemoglobin 116 g/l, reticulocytes 2.31% 0, platelets 162*10 9 /l, leukocytes 3.3*10 9 /l, neutrophils 65.9%. Investigation of PNH clone (D+157): PNH clone is not detected. The functioning of the graft is satisfactory, without the need for growth factors and replacement blood transfusions. Donor blood group. There are no manifestations of graft-versus-host disease, immunosuppressive therapy with cyclosporine A continues. R.M. Gorbacheva of St. Petersburg and hematologists of the city of Krasnoyarsk.

Discussion. Aplastic anemia and paroxysmal nocturnal hemoglobinuria are two progressive systemic diseases associated with high mortality. A large percentage of patients diagnosed with this pathology die within 5 years. At the age of 22, our patient was diagnosed with a super-severe form of AA with a large PNH clone, chronic intravascular hemolysis, which, according to the literature, is associated with a high percentage of lethal thrombotic complications. From the use of hemocomponent, as well as combined immunosuppressive therapy (thymoglobulin + cyclosporine A), no effect was observed in the patient. There was a pronounced anemic, hemorrhagic syndrome, deep pancytopenia in the hemogram, signs of intravascular hemolysis. The only possible treatment for this young patient in this situation was allogeneic BMT in combination with the use of pathogenetic therapy for PNH with a high-tech drug, eculizumab. In the department of TKM PSPbGMU them. Academician I.P. Pavlov Research Institute DogiT them. R.M. Gorbacheva, the patient was treated: the introduction of eculizumab 600 mg per day intravenously No. 3, followed by allogeneic BMT from a related donor, which ultimately led to the patient's recovery. The patient stopped anemic, hemorrhagic syndrome, normalized blood counts, signs of hemolysis disappeared, PNH-clone was not detected.

Thus, timely highly qualified diagnostics, a modern approach to the treatment of this disease, ensured a satisfactory outcome of the process in the patient, with an improvement in the clinical condition and a favorable prognosis for health.

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

The frequency of occurrence is 2 cases per 1 million people. The incidence is 1.3 cases per million people during the year. It is predominantly manifested in persons aged 25-45 years, the dependence of the incidence on gender and race has not been identified. There are isolated cases of the disease in children and adolescents.

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

Causes of the disease

Causes and risk factors for the development of the disease are unknown. It has been established that the pathology is caused by a mutation of 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) takes place in the red bone marrow. All specialized blood cells are formed from the so-called stem, non-specialized, cells that have retained the ability to divide. Formed as a result of successive divisions and transformations, mature blood cells enter the bloodstream.

Mutation of 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 the volume of the bone marrow, mutant cells multiply more actively than normal ones. In the hematopoietic tissue, a population of cells producing defective blood cells is rather quickly formed. At the same time, the mutant clone does not belong to malignant formations and can spontaneously disappear. The most active replacement of normal bone marrow cells by mutant ones occurs in the processes of bone marrow tissue recovery after significant lesions caused, in particular, by aplastic anemia.

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

Classification

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

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

The subclinical form of the disease is often preceded by aplastic anemia. There are no clinical manifestations of pathology, however, the presence of a small number of defective blood cells is detected only in laboratory studies.

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

The classical form proceeds with manifestations of typical symptoms, in the patient's blood there are populations of defective erythrocytes, platelets and some types of leukocytes. Laboratory research methods confirm intravascular destruction of pathologically altered cells, hematopoiesis disorders are not detected.

After the transferred diseases, leading to insufficiency of hematopoiesis, the third form of pathology develops. A pronounced clinical picture and intravascular lysis of erythrocytes develop against the background of bone marrow lesions.

There is an alternative classification, according to which they distinguish:

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

The severity of the course of the disease in different cases is not always interconnected with the number of defective erythrocytes. Both cases of a subclinical course with a content of modified cells approaching 90%, and extremely severe cases, with a replacement of 10% of the normal population, are described.

Development of the disease

At the moment, it is known that in the blood of patients with paroxysmal nocturnal hemoglobinuria, three types of erythrocytes with different sensitivity to destruction by the complement system can be present in the blood. In addition to normal cells, erythrocytes 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, pancytopenia is detected in patients - an insufficient content of blood cells of different types.

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

Pathology usually develops gradually, acute crisis onset is rare. Exacerbations are manifested against the background of menstruation, severe stress, acute viral diseases, surgical intervention, treatment with certain drugs (in particular, iron-containing ones). Sometimes the disease is exacerbated by the use of certain foods or for no apparent reason.

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

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

Outside the crisis, patients are concerned about manifestations of moderate general hypoxia, such as shortness of breath, attacks of arrhythmia, general weakness, and exercise tolerance worsens. During a crisis, abdominal pains are manifested, localized mainly in the navel, 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 pastosity of the face develops, yellowness of the skin and sclera is noticeable.

On a note! A typical symptom of the disease is urine staining. Approximately half of the known cases of the disease do not manifest themselves.

In 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. Perhaps the formation of blood clots in the vessels of the liver, kidneys, coronary and cerebral vessels are also affected, which can lead to death. Thrombosis localized in the vessels of the liver leads to an increase in the size of the organ. Violations of intrahepatic blood flow entail dystrophic tissue changes. With blockage of the portal vein system or the veins of the spleen, splenomegaly develops. Nitrogen metabolism disorders are accompanied by dysfunctions of smooth muscles, some patients complain of swallowing difficulties, spasms of the esophagus, erectile dysfunction is possible in men.

Important! Thrombotic complications in PNH predominantly affect the veins, arterial thrombosis is rare.

Video - Paroxysmal nocturnal hemoglobinuria

Mechanisms for the 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;
• urine staining 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 emerges 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 variant of the course of the disease is a crisis, interspersed with periods of a stable satisfactory state. In some patients, the periods between crises are very short, insufficient to restore blood composition. These patients develop persistent anemia. There is also a variant of the flow with an acute onset and frequent crises. Over time, crises become less frequent. In especially severe cases, a fatal outcome is possible, which is caused by acute renal failure or thrombosis of blood vessels that feed the heart or brain.

Important! Daily regularities in the development of hemolytic crises were not revealed.

In rare cases, the disease can also have a long calm course, isolated cases of recovery are described.

Diagnostics

In the early stages of the disease, diagnosis is difficult due to the manifestation of disparate nonspecific symptoms. It sometimes takes several months of observation to make a diagnosis. The classic symptom - specific staining of urine - appears during crises and not in all patients. Grounds for suspicion of Marchiafava-Micheli disease are:

• iron deficiency of unknown etiology;
• thrombosis, headaches, bouts 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 the transfusion of fresh donor blood.

In the process of diagnosis, it is important to establish the fact of chronic intravascular breakdown of erythrocytes and to identify specific serological signs of PNH.

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

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

Differential diagnosis with hemoglobinuria and anemia of a different etiology is necessary, in particular, autoimmune hemolytic anemia should be excluded. Common symptoms are anemia, jaundice, increased bilirubin in the blood. Enlargement of the liver or / and 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)	-	+
Ham test (acid)	-	+
Hemosiderin in urine	-	+
Thrombosis	±	+
Hepatomegaly	±	±
Splenomegaly	±	±

The results of the Hartman and Hem test are specific for PNH and are the most important diagnostic features.

Treatment

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

Attention! It is impossible to transfuse blood without preliminary preparation in such patients. Donor blood transfusion exacerbates the course of the crisis.

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

Additionally, folic acid, iron, hepatoprotectors are prescribed. With the development of thrombosis, direct-acting anticoagulants and heparin are used.

In extremely rare cases, the patient is shown 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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The materials are presented from the RUDN University textbook

anemia. Clinic, diagnosis and treatment / Stuklov N.I., Alpidovsky V.K., Ogurtsov P.P. - M .: LLC "Medical Information Agency", 2013. - 264 p.

Copying and replicating materials without specifying the authors is prohibited and punishable by law.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hemolytic anemia associated with a defect in the membrane of blood cells, therefore the disease is considered in the group of membranopathies and is the only acquired membranopathy among the diseases of this group. The mutation leading to the membrane defect in PNH occurs at the level of the pluripotent stem cell, and the cause of the mutation remains unclear.

PNH occurs with a frequency of 1:500,000 of the population. People of all age groups get sick, but more often - at the age of 30 - 40 years. Men and women get sick equally often.

Etiology and pathogenesis

Point mutation of a gene PIGA on chromosome 22 or the X chromosome of a pluripotent stem cell (PSC) leads to disruption of the formation of phosphatidylinolinic acid and proteins on the surface of blood cells CD 55 and CD 59, which form a system in normal cells that blocks the damaging effect on the membrane of activated complement due to the formation of a cascade CD 5b -9 - a complex that has a proteolytic effect on the cell membrane.

Thus, the absence of factors on the surface of blood cells that prevent complement function leads to the lysis of defective erythrocytes, neutrophils, and platelets.

With PNH, there are two clones in the blood of patients: normal and pathological, and the clinical picture and the severity of the disease largely depend on the ratio of these clones.

Clinic

The proteolytic action of activated complement leads to intravascular destruction of defective erythrocytes, which is manifested hemoglobinuria. Complement activation occurs at night during sleep, due to a shift in pH to the acid side.

Clinically, hemolysis during sleep is manifested by the release of black urine during morning diuresis, complaints of malaise, dizziness, and the appearance of yellowness of the sclera. In addition, hemolysis can provoke infectious diseases and certain drugs.

In addition to anemic symptoms associated with hemolysis, an important role in the PNH clinic is played by thrombotic complications, caused by the release of thromboplastin and a number of active enzymes from destroyed cells.

Often, one of the first complaints of the patient is abdominal pain, simulating a variety of acute abdominal pathology. Pain in the abdomen is associated with thrombosis of small mesenteric arteries.

Thrombophlebitisoccurs in 12% of patients with PNH and can proceed in different ways. In one of the options, the condition of patients outside of crises is quite satisfactory, the content Hb – about 80 – 90 g/l. In other patients, severe hemolytic crises follow one after another, leading to severe anemia. They are often accompanied by thrombotic complications.

Laboratory data

During a hemolytic crisis, there may be a sharp decrease in hemoglobin levels to 20 g / l and below, and a parallel decrease in the number of red blood cells. During the period of remission, the content Hb and erythrocytes increases, however, in rare cases it reaches the lower limit of the norm. Unlike most membranopathies, a defect in the erythrocyte membrane in PNH is not accompanied by characteristic changes in the shape of pathological erythrocytes. Anemia in most cases is normocytic and normochromic. However, with a significant loss of iron in the urine (as a result of hemoglobinuria and hemosiderinuria), erythrocyte hypochromia develops. The content of reticulocytes is increased, but to a much lesser extent than in congenital membranopathies with a similar intensity of hemolysis. Abnormal hemoglobins and a decrease in the activity of enzymes (except for acetylcholinesterase) in erythrocytes with PNH were not detected. Osmotic resistance of erythrocytes is not changed. During the incubation of erythrocytes of PNH patients under sterile conditions, autohemolysis is greater than normal, which, however, does not decrease when glucose is added.

The number of leukocytes in most cases is reduced due to neutropenia. Sometimes there is a left shift in the leukogram.

The number of platelets is usually also reduced. Platelet functions are not impaired.

When examining the bone marrow, erythroid hyperplasia and signs of insufficiency of bone marrow hematopoiesis are detected in the form of a violation of the maturation of red cells and granulocytic elements, as well as a decrease in the number of megakaryocytes, often with a violation of the lacing of platelets. In some patients with PNH, along with signs of dyshematopoiesis, bone marrow hypoplasia, characteristic of aplastic anemia, is found.

In cases where complement-sensitive PNH erythrocytes and symptoms of intravascular hemolysis are found in patients with previously established hematopoietic aplasia, PNH syndrome is diagnosed, which developed against the background of aplastic anemia.

However, one should be aware of the rare cases of PNH that end in aplastic anemia due to the depletion of bone marrow hematopoiesis by severe hemolytic crises and other adverse effects (infections, certain drugs, etc.).

An important laboratory sign of PNH is hemoglobinuria. The content of free hemoglobin in plasma due to intravascular destruction of erythrocytes in PNH, depending on the severity of hemolysis, ranges from 11 to 280 mg% (at a rate of up to 4 mg%).

The content of bilirubin is usually not sharply increased, mainly due to the unconjugated fraction. The level of serum iron in PNH depends on the phase of the disease: in hemolytic crises, due to the release of hemoglobin iron into the plasma, ferritinemia is observed, and in the period of a calm course, due to the loss of iron in the urine, hypoferritinemia is observed. Iron deficiency in PNH, in contrast to iron deficiency anemia, is accompanied by a simultaneous decrease in total and latent iron-binding capacity, apparently due to impaired transferrin synthesis in the liver.

In the study of urine in most patients with PNH, hemoglobinuria is detected. With PNH, hemoglobin appears in the urine at a relatively low concentration in plasma, which is associated with a decrease in the content of plasma haptoglobin. During the excretion of hemoglobin by the kidneys, part of it is reabsorbed and deposited in the epithelium of the tubules in the form of hemosiderin, which is then excreted in the urine. Interestingly, hemosiderinuria in PNH can be caught more often than hemoglobinuria, since it also develops outside the hemolytic crisis.

DiagnosticsThe disease is associated with the identification of a characteristic clinical picture, laboratory signs of intravascular hemolysis (hemoglobinemia (red color of blood serum after centrifugation), a decrease in haptoglobin to the blood, a slight indirect bilirubinemia, an increase in LDH, hemoglobinuria, hemosiderinuria). The diagnosis of PNH is based on the detection of complement-sensitive erythrocytes characteristic of this disease. For this purpose are used Hem's acid test and more sensitive sucrose test.

When staging the Hema test, the studied erythrocytes are incubated in normal serum acidified to pH 6.4. Under these conditions, only complement-sensitive erythrocytes are lysed. It should be remembered that with a small content of PNH-erythrocytes in the patient's blood and with low complement activity in serum, the Hem test may give negative results.

More sensitive is the sucrose test, in which the studied erythrocytes and a small amount of normal serum are placed in an isotonic sucrose solution. Under conditions of reduced voltage in a sucrose medium, more active fixation of complement on the surface of erythrocytes and lysis of complement-sensitive PNH erythrocytes occur.

The evidence for the presence of a PNH clone is the detection on the cell membrane of signs characteristic of PIG A gene damage. hemolysis. The most reliable is the study of monocyte granulocytes, since nucleated cells are less susceptible to the action of complement.

Treatment

Due to the lack of clear ideas about the pathogenesis of PNH, the treatment of this disease is currently symptomatic.

In order to combat anemia, replacement blood transfusions are used, the frequency of which depends on the severity of hemolysis and the compensatory activity of the bone marrow. It should be remembered that the transfusion of fresh whole blood in patients with PNH is often accompanied by an increase in hemolysis. The reason for this reaction is unclear. Patients with PNH better tolerate transfusions of whole blood or erythrocyte mass of long-term storage (more than 7–8 days) and transfusions of 3–5 times washed erythrocytes freed from leukocytes and platelets. The use of washed erythrocytes is the best transfusiological method in the treatment of PNH. When a reaction to washed erythrocytes also appears due to the development of isosensitization, an individual selection of a donor is necessary according to the indirect Coombs reaction (Fig. 12).

An important place in the treatment of PNH is occupied by iron preparations and androgenic hormones. Therapy with iron preparations is recommended for patients with PNH if erythrocyte hypochromia and a decrease in serum iron levels are detected during the calm course of the disease. Iron preparations should be used carefully (in small doses and only peros ), since their ability to provoke severe hemolytic crises in some patients with PNH is known.

The use of androgens in PNH is based on the stimulating effect of these hormones on erythropoiesis. The appointment of Nerabol or its analogues at a dose of 30-40 mg / day contributes to a more rapid recovery of hemoglobin levels after a hemolytic episode and thereby significantly reduces the need for blood transfusions. Especially effective is the use of androgens in PNH with hematopoietic hypoplasia.

The tactics of treating thrombotic complications depends on the localization of thrombosis, their duration and the state of the coagulation system. In cases where this complication threatens the life of the patient, it is necessary to use complex thrombolytic and anticoagulant therapy (fibrinolysin or urokinase, nicotinic acid, heparin and indirect anticoagulants) according to general therapeutic rules and in sufficient dosages.

Since there are reports of increased hemolysis after the administration of heparin, this anticoagulant should be used with great caution.

Splenectomy for PNH is not indicated, since the postoperative period is often complicated by thrombosis of the mesenteric vessels. The risk of surgery is acceptable only if there are pronounced symptoms of hypersplenism: deep leukopenia complicated by frequent infections and / or thrombocytopenia, accompanied by severe hemorrhagic syndrome.

A modern genetically engineered drug Eculizumab (eculizumab) (SOLIRIS®) has been developed, which is registered by the FDA (Food and Drug administration) for the treatment of children and adults suffering from PNH. Eculizumab is a glycosylated humanized monoclonal antibody, kappa-immunoglobulin (IgG2/4k), that binds to human complement protein C5 and inhibits the activation of complement-mediated cell lysis. The antibody consists of constant regions of human immunoglobulin and complementary-deterministic regions of mouse immunoglobulin embedded in the variable regions of the light and heavy chains of a human antibody. Eculizumab contains two heavy chains of 448 amino acids each and two light chains of 214 amino acids each. The molecular weight is 147870 Da. Eculizumab is produced in cultured mouse myeloma NS0 cells and purified by affinity and ion exchange chromatography. In the production process of the substance, the processes of specific inactivation and removal of viruses are also included.

Eculizumab inhibits the terminal activity of human complement, having a high affinity for its C5 component. As a result, the splitting of the C5 component into C5a and C5b and the formation of the terminal complement complex C5b–9 are completely blocked. Thus, eculizumab restores the regulation of complement activity in the blood and prevents intravascular hemolysis in patients with PNH. On the other hand, terminal complement deficiency is accompanied by an increased incidence of infections with encapsulated microorganisms, mainly meningococcal infections. At the same time, eculizumab maintains the content of early complement activation products necessary for opsonization of microorganisms and elimination of immune complexes. Appointment to patients of the drug Soliris is accompanied by a rapid and stable decrease in terminal complement activity. In most patients with PNH, a plasma concentration of eculizumab of about 35 μg / ml is sufficient to completely inhibit intravascular hemolysis induced by terminal complement activation.

Due to the unique new clinical results and the opening therapeutic opportunities for physicians to preserve the full life and health of patients, Eculizumab was registered in an accelerated manner, without conducting the third phase of clinical trials - this will save many lives, both children and adults.

In this regard, following the registration in the US, the European Medicines Committee issued a positive opinion on the accelerated registration of Eculizumab in Europe, which is also expected in the near future.

Given the high cost of eculizumab, its inability to target the cause of the disease, and the lifelong use of eculizumab, it is most suitable for a reserve strategy designed specifically for patients with high PNH cell counts or for patients with a tendency to thrombosis, not depending on the size of the PNH clone.

Currently, the only curative treatment for PNH is allogeneic bone marrow transplantation.

Course and forecast

The prognosis depends on the severity of the course of the underlying disease, worse in patients dependent on blood transfusions, with severe thrombosis. In 10% of patients, spontaneous remissions of the disease are observed, in others, transformation into aplastic anemia, MDS, in 5% - into acute leukemia. The average life expectancy is 10-15 years.

PNH is a chronic and currently still completely incurable disease. The severity of PNH and prognosis largely depend on the size of the complement-sensitive erythrocyte population, the compensatory capacity of the bone marrow, and the occurrence of complications, especially venous thrombosis. The idea of ​​a severe prognosis in PNH has changed significantly in recent years due to the introduction of active symptomatic therapy.

The number of patients who are in a state of clinical and hematological compensation for a long time and lead a normal life at this time has increased. The incidence of severe, life-threatening thrombosis has decreased. In some patients, over time, there is a softening of the course of the disease with a decrease in the proportion of complement-sensitive erythrocytes. In rare cases, the complete disappearance of pathological erythrocytes is described, which indicates the fundamental possibility of curing the disease.

Aplastic anemia is a rare disease of the blood system characterized by pancytopenia in the peripheral blood and hypocellular (up to complete aplasia) bone marrow with the replacement of active hematopoietic tissue with adipose tissue. The first description of the disease, made by P. Ehrlich, refers to 1888.

The disease occurs in most regions of Europe and America with a frequency of 2-3 cases per year per 1 million population. The incidence of aplastic anemia is 2-3 times higher in East Asia. Two peaks of incidence are noted: at the age of 10 to 25 years and in persons over 60 years of age without significant differences by sex. A rare form is congenital aplastic anemia - Fanconi anemia, which in most cases manifests itself as an autosomal recessive disease.

Etiology and pathogenesis
The etiology of the disease in 70-80% of cases is unknown (idiopathic forms), and in other cases, the occurrence of aplastic anemia is associated with various chemical, physical factors, infections (post-hepatitis aplastic anemia, forms associated with cytomegalovirus, parvovirus infection, etc.).

The most common are acquired forms of aplastic anemia, but up to 15-20% of cases of the disease can be constitutional / congenital variants (Fanconi anemia, anemia associated with dyskeratosis), accompanied by various cytogenetic anomalies. There is also a variant of aplastic anemia associated with paroxysmal nocturnal hemoglobinuria.

The main pathogenetic mechanism for the development of hematopoietic aplasia in aplastic anemia is immune-mediated damage to the hematopoietic stem cell. At the same time, a functional defect in hematopoietic stem cells and pathology of the hematopoietic microenvironment are not excluded.

Evidence of active immune processes in the bone marrow of patients with aplastic anemia is an increase in the content of mature and activated T-lymphocytes, cells with the suppressor-killer phenotype, inversion of the helper-suppressor ratio, naturally detected in this group of patients.

Characterized by an increase in the level of cytokines that negatively affect the processes of hematopoiesis, such as IFNu, IL-2, tumor necrosis factor (TNFα). At the same time, apparently, an enhanced uncontrolled trigger mechanism of Fas-dependent apoptosis of hematopoietic cells also plays a significant role in the development of the disease. For patients with aplastic anemia, a deficiency of factors that regulate hematopoiesis is usually not characteristic. There are certain pathogenetic relationships between aplastic anemia, paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome, the nature of which is still not entirely clear. Aplastic anemia can eventually transform into paroxysmal nocturnal hemoglobinuria and myelodysplastic syndrome. PNH-clone of small size without signs of hemolysis is detected, according to recent studies, in 50-70% of patients with aplastic anemia. Clones with cytogenetic abnormalities, in the absence of evidence in favor of myelodysplastic syndrome, can be determined in some patients with aplastic anemia.

Clinical picture
According to the severity of the course, high early mortality among patients with severe aplastic anemia and the complexity of treatment, this category is comparable to the group of patients with acute leukemia. Mortality without treatment in the first 6 months in severe forms of aplastic anemia reaches 50% or more. The causes of death of patients are the progression of the disease and the development of hemorrhagic and severe infectious complications.

Clinical manifestations of the disease are mainly due to the presence of anemic and hemorrhagic syndrome. Patients with aplastic anemia are characterized by varying degrees of pallor of the skin and visible mucous membranes. As a rule, on the skin and mucous membranes there are hemorrhages of various sizes - from small punctures to confluent ones. Often there are hemorrhages in the fundus of the eye, the retina, which is accompanied by a decrease in visual acuity. Hemorrhages on the mucous membranes of the oral cavity may be accompanied by symptoms of stomatitis, soft tissue necrosis. In severe forms of the disease with severe hemorrhagic manifestations, hemorrhages in the intestinal wall are possible. In the latter case, there will be a corresponding clinical picture: pain syndrome, bloating and tenderness on palpation, disturbances in peristalsis. At the same time, in some patients (up to 20% on average), no visible hemorrhagic manifestations are noted during the initial examination. Changes in the cardiovascular system are manifested by tachycardia, expansion of the boundaries of the heart, muffled heart tones, systolic murmur over the surface of the heart.

Lymphadenopathy, hepato- and splenomegaly are not typical for aplastic anemia. With deep granulocytopenia, there is an increased tendency to develop infectious and inflammatory-necrotic complications.

Acute onset in aplastic anemia is observed in 12-15% of patients and is accompanied by fever, necrotic tonsillitis, severe nasal, gingival, uterine bleeding, the appearance of multiple hemorrhages on the skin and mucous membranes. In more than 80% of patients, the disease develops gradually with increasing manifestations of anemic and hemorrhagic syndrome.

With Fanconi anemia, usually detected at a young age, skeletal anomalies can be determined, skin pigmentation - spots of the color "coffee with milk".

Laboratory research
Complete blood count usually shows pancytopenia with relatively intact lymphocytes. Anemia is usually normochromic and is characterized by reticulocytopenia. Macrocytosis may be noted. Platelets are greatly reduced in number and are usually small.

The picture of the bone marrow of patients with aplastic anemia is characterized by a reduced number of hematopoietic cells and enlarged fatty spaces. Erythropoiesis is narrowed or absent, dyserythropoiesis is often noted, not accompanied by dysplastic changes in other rows of hematopoiesis, as in myelodysplastic syndrome. The number of megakaryocytes and granulocytic cells is significantly reduced. Since bone marrow damage is uneven, focal hyperplasia of erythroid and granulocyte sprouts can be observed, and when their “hot pocket” with a focus of intact hematopoiesis is aspirated, myelogram parameters, especially in the early stages of the disease, can be close to normal. To assess the overall cellularity and assess the morphology of residual hematopoietic cells, the study of a high-quality bone marrow trephine biopsy specimen is of decisive importance.

Differential Diagnosis
The diagnosis of aplastic anemia is based on the determination of pancytopenia in the peripheral blood and reduced cellularity of the bone marrow according to trephine biopsy. The replacement of active hematopoietic tissue with adipose tissue is characteristic, in the absence of infiltration by atypical cells and signs of fibrosis. Careful examination of blood smears and bone marrow preparations allows us to exclude the presence of dysplastic neutrophils and abnormal platelets, tumor cells.

International research groups have recommended that the diagnosis of aplastic anemia be based on the presence of at least two of the following blood parameters in combination with characteristic changes in the bone marrow picture: hemoglobin level
The plan for examining patients with suspected aplastic anemia includes a complete clinical blood test with the determination of the number of platelets and reticulocytes, myelogram count and histological examination of bone marrow trephine biopsy. In order to identify variants of the disease associated with the presence of a PNH clone, all patients with aplastic anemia should be tested for paroxysmal nocturnal hemoglobinuria using highly sensitive flow cytometry. Potential bone marrow recipients undergo HLA-typing of blood cells.

For the diagnosis of rare congenital forms of the disease, a thorough history taking and examination of the patient is important. In order to exclude Fanconi anemia, a chromosomal analysis of blood lymphocytes is indicated - a test for induced chromosomal breakdowns with diepoxybutane or mitomycin.

When conducting differential diagnosis, it is necessary to exclude cytopenias of secondary origin. This, in addition to a detailed history and examination, may require tests such as determining the level of vitamin B12 and folates in the blood, testing for viruses, immunophenotyping of bone marrow cells, ultrasound and echocardiography, tests to rule out rheumatoid diseases and other tests as indicated.

Differential diagnosis is also carried out with acquired partial red cell aplasia and a congenital form - Diamond-Blackfan anemia, in which aplasia of the erythroid germ of the bone marrow is detected with the preservation of granulo- and thrombocytopoiesis.

Classification
To determine the tactics of therapy, it is necessary to determine the severity of aplastic anemia. In accordance with the international classification, it is customary to distinguish between severe and non-severe forms of aplastic anemia. The main purpose of this classification was to identify a group of patients who are primarily indicated for bone marrow transplantation because of the risk of early death.

Treatment
The strategy for the treatment of aplastic anemia should be aimed at restoring the deficiency of hematopoietic stem cells and suppressing destructive immunological processes.

Complete restoration of bone marrow hematopoiesis in patients with aplastic anemia can only be achieved with hematopoietic stem cell transplantation, which is the method of choice in young patients with severe and super-severe forms of the disease. However, the main method of therapy for most patients is immunosuppressive therapy, as it is more affordable, with fewer contraindications and is comparable to hematopoietic stem cell transplantation in terms of effectiveness.

The first attempts to treat aplastic anemia with bone marrow transplants were made as early as the 1930s, but the complexity and imperfection of the technology for selecting donors and the methods of transplantation at that time limited the possibilities of using transplantation. With the improvement of donor selection technology and technique, bone marrow transplantation has entered the standard of care for patients with severe aplastic anemia as a method of choice in newly diagnosed patients with severe aplastic anemia in the presence of an HLA-identical related donor and as a method of therapy for patients with severe disease who did not respond. for treatment with antithymocyte immunoglobulin and cyclosporine. An increase in the efficiency of allogeneic bone marrow transplantation was achieved as a result of a decrease in the frequency of infectious complications, improvement of pre-transplant preparation regimens, a decrease in the incidence of rejection reactions and graft-versus-host disease.

According to the European Working Group for the study of bone marrow transplantation and aplastic anemia, the survival rate of patients with severe aplastic anemia after hematopoietic stem cell transplantation, which was in 1970-1979. 43%, in 1991-1996 increased to 69%, and by 1997-2002. - up to 72%. Long-term survival of patients with aplastic anemia after transplantation can currently reach 80-96%. The preferred source of hematopoietic stem cells for patients with aplastic anemia is bone marrow.

Patients with non-severe aplastic anemia and severe aplastic anemia over the age of 40 years and/or without an HLA-matched sibling donor are recommended to undergo a course of immunosuppressive therapy. The use of immunosuppressive therapy is based on the concept of the pathogenesis of aplastic anemia as a pathological process caused by impaired immune regulation of hematopoiesis. The standard regimen of immunosuppressive therapy, which gives the best results for patients with both severe aplastic anemia and non-severe aplastic anemia, is a combination of antithymocyte immunoglobulin and cyclosporine A. The benefits of combination therapy have been confirmed by many research groups. Thus, the 11-year results of immunosuppressive therapy, according to the German group of scientists, showed an increase in the frequency of remissions when antithymocyte immunoglobulin and cyclosporine were added to therapy from 41 to 70% in the general group of patients and from 31 to 65% in severe aplastic anemia. At the same time, the median time to achieve remission decreased from 82 to 60 days, and relapse-free morbidity increased by 18%.

Antithymocyte immunoglobulin is a drug obtained by immunizing animals with human lymphocytes (fetal thymocytes). The drugs of this series have a selective lymphocytotoxic effect on activated T-suppressors, inhibit the production of suppressive cytokines by T-cells, act on apoptosis by reducing Fas-antigen expression on the CD+ cells of the bone marrow of patients.

Cyclosporin A - a metabolite of the fungus Tolipocladium inflatum, a cyclic polypeptide that selectively and reversibly changes the function of lymphocytes, inhibiting the production and fixation of lymphokines on specific receptors; inhibits the G0 and G1 phases of the cell cycle of immunocompetent cells, reduces the activity of genes responsible for the synthesis of IL-2 and a number of other cytokines. The advantage of CsA is its specific reversible action in the absence of an overwhelming effect on hematopoiesis, as well as the relative preservation of anti-infective immunity.

Courses of therapy with antithymocyte immunoglobulin, lasting 4-5 days, are carried out in a hospital. The recommended doses of the drug for equine antithymocyte immunoglobulin are 20-40 mg/kg of body weight. To improve the results and prevent allergic reactions, serum sickness, glucocorticoids are usually prescribed simultaneously in the form of a short course [methylprednisolone at a dose of 1-3 mg / kg)]. At the end of the administration of antithymocyte immunoglobulin for a long (from 6 months) period, oral CsA preparations are prescribed at doses of 5-7 mg/kg and higher in the absence of significant toxicity. When using this mode, the response rate is 60-80% with a 5-year survival rate of patients with severe aplastic anemia of 75-85%.

The first persistent positive results during immunosuppressive therapy are usually observed after 2-3 months, and therefore it is advisable to determine the results of therapy after 3-6 months from the start of treatment. The criteria for the effectiveness of therapy are complete and partial remission. Complete clinical and hematological remission implies the absence of clinical symptoms of the disease, complete relief of hemorrhagic syndrome, hemoglobin content of more than 110 g/l; the content of granulocytes is more than 1.0x109 / l, platelets is more than 100x109 / l (in other cases - more than 125-150x109 / l). Partial clinical and hematological remission is characterized by the absence of clinical symptoms of the disease and manifestations of hemorrhagic syndrome, hemoglobin content of more than 80 g/l with independence from hemocomponent therapy, granulocyte content of more than 0.5x109/l, platelets more than 20.0x109/l.

Clinical and hematological improvement may also be a positive result, in which there are no pronounced hemorrhagic manifestations, the need for hemocomponent therapy is reduced, and hematological parameters improve with a granulocyte content of more than 0.5x109/l, platelets more than 20.0x109/l.

To assess the effectiveness of the treatment of patients with aplastic anemia, depending on the severity of the course of the disease, the European group of experts proposes the following criteria. According to current recommendations, CsA should be continued after receiving the maximum hematological response [persistent partial remission with improvement in all hematopoietic lineages, complete remission] from 6 to 12 months, followed by gradual withdrawal, which minimizes the number of relapses.

There is a positive experience with the use of high doses of cyclophosphamide in the 1st line of therapy. The first publications dating back to 1996 showed a good effect of immunosuppressive therapy with these drugs in patients with aplastic anemia, but in the presence of serious complications during therapy, including fatal infections. However, as adjuvant therapy improves, more recent publications show good treatment outcomes with more complete and sustained remissions in patients with severe aplastic anemia, although these results have not been confirmed by randomized controlled trials.

If the first course of combination therapy with immunoglobulin antithymocyte/CsA is ineffective for patients with severe aplastic anemia, the possibility of bone marrow transplantation from a compatible unrelated donor is considered. At the same time, the probability of favorable results is higher when transplantation is performed at an earlier date.

The disadvantages of immunosuppressive therapy as a method of treating patients with aplastic anemia include:
preservation of residual defects in hematopoiesis (in the form of preservation of foci of bone marrow hypoplasia, functional inferiority of myelokaryocytes);
high risk of recurrence (up to 20-30% of patients and above);
late clonal complications (up to 20-60% with long-term follow-up), including myelodysplastic syndrome, acute leukemia, paroxysmal nocturnal hemoglobinuria.

The frequency of relapses in patients with aplastic anemia after the 1st line of immunosuppressive therapy is relatively high, however, in most cases, such relapses are successfully treated with repeated courses of immunosuppressive therapy and do not significantly worsen the overall prognosis. Thus, recent studies have shown that in case of relapse after the first successful course of therapy, which included antithymocyte immunoglobulin, repeated courses lead to remissions in 11-65% of patients.

In the 2nd and subsequent lines of therapy, it is possible to use drugs such as alemtuzumab, mycophenolic acid preparations for CsA intolerance. There is evidence of a positive experience with the use of daclizumab (recombinant monoclonal antibodies against the IL-2 receptor) and a number of other immunosuppressive drugs, but there is still no sufficiently convincing data on their use in large groups of patients with aplastic anemia.

Splenectomy, previously used in the treatment of patients with aplastic anemia, is now rarely used, although some authors consider its use justified in the 2nd-3rd line of therapy, especially in the presence of an autoimmune component.

It has been shown that in improving the results of therapy for aplastic anemia, the early start of a course of treatment and adequate accompanying therapy are of great importance. The latter includes hemocomponent replacement therapy to maintain the level of red blood cells and platelets at a safe level.

Indications for the appointment of thromboconcentrate is hemorrhagic syndrome with platelets
In recent years, attempts have been made to use thrombopoietin receptor agonists (eltrombopag) to control hemorrhagic syndrome, with good results. Moreover, there are data indicating the ability of thrombopoietin receptor agonists to lead not only to an increase in the number of platelets and relief of hemorrhagic manifestations, but also to improvement in other cell lines.

Since transfusion dependence often leads to post-transfusion iron overload in patients with aplastic anemia, patients with frequent red blood cell transfusions and serum ferritin levels above 1000 mg/l are treated with iron chelators.

If infectious complications occur in patients with aplastic anemia, therapy is carried out according to the rules common for patients receiving immunosuppressive therapy, with the appointment of broad-spectrum antibacterial drugs, antifungal drugs according to indications.

The use of hematopoietic stimulants - granulocyte colony-stimulating factor and erythropoietin - in patients with aplastic anemia is not considered expedient by most researchers because of their low effectiveness in this group of patients and an increased risk of developing clonal complications. Data from long-term observations and meta-analysis, regularly presented during the 2000s at scientific congresses and hematological conferences of the American Society of Hematology, the European Hematology Association, the European Group for Bone Marrow Transplantation and others, showed that the use of erythropoietin and G-CSF does not significantly affect reduction in mortality or increase in complete and overall responses to therapy. Nevertheless, short courses of G-CSF may be recommended for severe systemic infections in patients with profound granulocytopenia. The prognosis of the disease depends mainly on the severity of aplasia and early initiation of active therapy. Without treatment, in severe forms, up to 50% of patients die in the first months, and with modern therapy, long-term survival is 70-80%.

With regard to immunosuppressive therapy, the results of treatment are better in patients with an early granulocytic and reticulocyte response. Available data also indicate a better response to immunosuppressive therapy in patients with aplastic anemia associated with the presence of a PNH clone. Among the factors influencing the prognosis of the disease are the effectiveness of immunosuppressive therapy and the likelihood of clonal evolution; recently, attention has been paid to the shortening of the length of telomeres in blood cells.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disease characterized 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 out of half a million, mostly young people.

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

Symptoms of PNH outside the crisis:

• general weakness;
• pale, with an icteric shade coloring of an integument;
• anemia;
• tendency to thrombosis; hematuria; high blood pressure; liver enlargement; dyspnea; heartbeat; frequent infectious diseases.

Diagnostics

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

Treatment of paroxysmal nocturnal hemoglobinuria

The 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 administration of antithymocyte immunoglobulin at a dose of 150 mg / day, for 4 to 10 days. It is recommended to take iron preparations per os in small dosages. Sometimes corticosteroids in high dosages give a good effect. Bone marrow hypoplasia with the development of thrombotic complications are indications for bone marrow 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.