Paroxysmal nocturnal hemoglobinuria: clinical guidelines

List of Abbreviations

PNH — paroxysmal nocturnal hemoglobinuria

AA — aplastic anemia

LDH – lactate dehydrogenase

MDS — myelodysplastic syndrome

HSC — hematopoietic stem cell

MAC — membrane attack complex

GPI – glycosylphosphatidylinositol

mPAP – mean pulmonary arterial pressure

CKD – chronic kidney disease

ULN – upper limit of normal

GC – glucocorticoids

HSCT – hematopoietic stem cell transplantation.

Terms and Definitions

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, bone marrow failure, and an increased risk of thrombotic and organ complications.

Intravascular hemolysis is the process of autologous erythrocyte destruction within the vascular bed, driven primarily by complement-mediated mechanisms and associated with the release of large amounts of free hemoglobin into blood plasma.

Hemolytic crisis is a sharp exacerbation of the clinical manifestations of PNH, a decrease in hemoglobin and red blood cell counts, accompanied by an elevation in laboratory markers of active hemolysis (indirect bilirubin, LDH activity, reticulocyte count), in most cases associated with prior exposure to triggering factors such as infections, certain medications, etc.

Breakthrough hemolysis is the loss of intravascular hemolysis control, including the development of a hemolytic crisis, during ongoing PNH disease-modifying therapy. Pharmacokinetic breakthrough hemolysis develops in patients with individual pharmacokinetic parameters resulting in decreased eculizumab** concentrations 1–4 days prior to the next scheduled dose in the maintenance phase. Pharmacodynamic breakthrough hemolysis is associated with complement-amplifying conditions (infections, surgery, pregnancy, etc.) and can develop in any patient.

PNH clone refers to the progeny cells of a hematopoietic stem cell with a mutation in the PIGA gene, which disrupts the expression of membrane proteins, including those that protect cells from complement-mediated destruction.

1. Brief information on the disease or condition

1.1 Definition of the disease or condition

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, bone marrow failure, and an increased risk of thrombotic and organ complications.

1.2 Etiology and pathogenesis of the disease or condition (group of diseases or conditions)

The pathogenesis of PNH is based on complement-mediated erythrocyte lysis resulting from an acquired genetic defect that leads to impaired expression of critical regulatory glycoproteins on the cell surface.

Genetic mutations

According to the modern concept of PNH pathogenesis, a somatic mutation of the PIGA gene located on the X chromosome occurs in a hematopoietic stem cell (HSC) [1,2]. This acquired mutation disrupts the biosynthesis of glycosylphosphatidylinositol (GPI), a glycolipid structure that serves as an "anchor" for attaching numerous glycoproteins to the cell surface, including those that regulate complement activity. This results in the complete or partial loss of surface GPI-linked proteins—CD55 and CD59—on the progeny cells of the mutant HSC, which normally protect cells from complement-mediated lysis [1–5].

A somatic mutation in the PIGA gene can occur in one or several HSCs. Subsequently, mutant HSCs gain a clonal advantage over normal cells, leading to the development and manifestation of PNH. The putative mechanisms of pathological HSC expansion include clonal selection and clonal evolution.

Complement system and the role of surface glycoproteins CD55 and CD59

The complement system is an essential component of innate immunity, playing a key role in defense against infections and the elimination of damaged autologous cells. Under normal conditions, complement activity is strictly regulated by a number of soluble proteins as well as cell-surface glycoproteins, including DAF (CD55) and MIRL (CD59). The complement system is activated via three distinct pathways: classical, lectin, and alternative. Despite differences in the triggers required to activate each complement pathway, all pathways culminate in a cascade of reactions leading to the formation of C3 convertase, which cleaves the C3 component into two fragments: C3a, a pro-inflammatory anaphylatoxin, and C3b. The functions of the C3b component include opsonization of pathogens and defective autologous cells, as well as activation of the next stage of the terminal complement cascade. The presence of C3b is required for the formation of C5 convertase, which cleaves the C5 complement component into C5a—a pro-inflammatory mediator—and C5b, which initiates the assembly of the membrane attack complex (MAC) on the cell surface [6]. Once assembly is complete, the MAC forms a transmembrane channel in the cell, leading to cell death [7,8]. Normally, C3 convertase activity is regulated by the GPI-anchored glycoprotein CD55, while MAC assembly on the cell surface is blocked by another GPI-anchored inhibitor, CD59 [9–11]. In the absence of CD55 and CD59 on the cell surface, uncontrolled activation of complement components and MAC assembly occur, followed by cell lysis [12].

Coagulation system in PNH

The mechanisms underlying the marked increase in thrombogenicity in PNH are not yet fully understood [13]. During hemolysis, large amounts of erythrocyte breakdown products are released into the vascular bed, including free hemoglobin, which is capable of inducing platelet activation, depleting nitric oxide stores, and leading to local thrombophlebitis [14–18]. All of these factors are associated with an increased risk of thrombosis. In addition to the prothrombotic effects of red blood cell breakdown products, there are also direct interactions between the complement and coagulation systems: the C5a component can induce leukocyte expression of tissue factor and plasminogen activator inhibitor type 1 [19,20].

1.3 Epidemiology of the Disease or Condition

PNH is an extremely rare disease. According to the international PNH Registry, initiated in 2003, the incidence of PNH is 1–1.5 per 1,000,000 population per year [21,22]. The prevalence is 1.59 per 100,000 individuals [23].

1.4 Features of Coding the Disease or Condition

According to ICD-10, paroxysmal nocturnal hemoglobinuria corresponds to code D59.5 "Paroxysmal nocturnal hemoglobinuria".

1.5 Classification of the disease or condition

According to ICD-10, paroxysmal nocturnal hemoglobinuria corresponds to code D59.5 "Paroxysmal nocturnal hemoglobinuria".

1.6 Clinical presentation of the disease or condition

In most cases, PNH develops gradually as slowly progressive weakness, fatigue, episodes of jaundice, and dark urine, sometimes accompanied by low-grade fever. A marked deterioration in condition may occur during a hemolytic crisis [24].

It is practical to divide the clinical manifestations of PNH into several categories:

1) Symptoms caused by hemolytic anemia.

Patients complain of fatigue, shortness of breath, tachycardia, jaundice of the skin and mucous membranes, and dark urine (ranging up to black). The condition may significantly worsen during hemolytic crises, the onset of which is often triggered by infections.

2) Symptoms caused by thrombotic complications.

Thrombosis is the most common cause of death in patients with PNH. Venous thrombosis occurs somewhat more frequently than arterial thrombosis and is characterized by unusual locations: thrombosis of the hepatic veins (Budd-Chiari syndrome), portal system vessels, and cerebral venous sinuses. Symptoms in these cases are determined by the site of thrombosis.

3) Symptoms caused by smooth muscle dystonia.

Presumably, this category of symptoms is caused by the depletion of the nitric oxide (NO) pool associated with elevated plasma concentrations of free hemoglobin. The most characteristic clinical manifestations of smooth muscle dystonia in PNH patients include dysphagia, esophageal spasm, abdominal pain, and erectile dysfunction.

4) Other symptoms.

Patients with PNH have a 6-fold higher risk of developing chronic kidney disease (CKD), which may be caused by microvascular renal thrombosis as well as the damaging effects of iron deposits accumulating in the renal parenchyma as a result of erythrocyte destruction [25].

Approximately 1/3 of PNH patients not receiving disease-modifying therapy develop symptoms of pulmonary hypertension. The main complaint of patients with pulmonary hypertension is shortness of breath. Probable causes of pulmonary hypertension include both pulmonary arteriolar spasm resulting from nitric oxide pool depletion due to active hemolysis and chronic thrombosis of small branches of the pulmonary artery [26].

2. Diagnostics, medical indications and contraindications for diagnostic methods

Criteria for establishing the diagnosis/condition: the diagnosis of PNH is established based on:

• characteristic clinical and laboratory findings of intravascular hemolysis

• detection of a PNH clone in peripheral blood by flow cytometry (peripheral blood cell immunophenotyping for the diagnosis of paroxysmal nocturnal hemoglobinuria using an expanded marker panel, including FLAER (fluorescently labeled aerolysin)

2.1 Complaints and Anamnesis

See the "Clinical Presentation" section

2.2 Physical Examination

See the "Clinical Presentation" section

2.3 Laboratory Diagnostics

The gold standard for diagnosing PNH in patients presenting with clinical and laboratory features of intravascular hemolysis is the detection of GPI-anchored glycoproteins on the surface of erythrocytes (CD59), neutrophils (CD24/FLAER), and monocytes (CD14/FLAER) using flow cytometry (peripheral blood cell immunophenotyping with an expanded marker panel including FLAER (fluorescently labeled aerolysin) for the diagnosis of paroxysmal nocturnal hemoglobinuria. Previously used tests, such as the Ham test (acid hemolysis test) and sucrose hemolysis test, are now of historical significance only and are not recommended for clinical use.

2.3.1 Diagnosis of anemia and intravascular hemolysis

- complete blood count (CBC) with differential;

- blood reticulocyte count;

- routine biochemical blood test (determination of alanine aminotransferase, aspartate aminotransferase, LDH, creatinine, urea, conjugated (direct) bilirubin, unconjugated (indirect) bilirubin, ferritin, total iron-binding capacity, serum iron, plasma free hemoglobin, and haptoglobin if available);

- direct antiglobulin test (direct Coombs test);

- indirect antiglobulin test (indirect Coombs test) — only for patients receiving red blood cell transfusions;

- complete urinalysis with determination of urinary hemosiderin.

GCP CLEL 5Grade of Recommendation – C (Level of Evidence – 5)

Comments: active intravascular hemolysis is characterized by elevated LDH, increased total bilirubin due to the indirect fraction, elevated plasma free haptoglobin depletion, and the presence of hemosiderin in the urine.

2.3.2 Determination of the PNH clone

Comments: PNH should be suspected in patients presenting with the following clinical and laboratory manifestations:

• Presence of signs of intravascular hemolysis

• Presence of signs of hemolysis combined with:

• Hypochromic and/or microcytic anemia

• Abdominal pain

• Dysphagia

• Thrombosis

• Neutropenia or thrombocytopenia

• Thrombosis in rare sites or with unusual presentations:

• Hepatic vein thrombosis (Budd-Chiari syndrome), portal vein thrombosis, cerebral venous sinus thrombosis, dermal vein thrombosis)

• Concomitant hemolytic anemia

• Concomitant unexplained cytopenia

• Signs of bone marrow failure

• Suspected or confirmed aplastic anemia

• Suspected or confirmed MDS

• Cytopenic syndromes of unexplained etiology following a comprehensive hematological workup.

Flow cytometry detects the percentage of cells with a deficiency or total absence of GPI-anchored glycoproteins (GPI-AP). Three types of erythrocytes are distinguished: erythrocytes with normal GPI-AP expression (Type I), erythrocytes with partial GPI-AP deficiency (Type II), and erythrocytes with complete absence of GPI-AP (Type III). The laboratory reports the percentage of each cell type in the peripheral blood. Flow cytometric analysis of the PNH clone must follow the 2018 recommendations of the International Clinical Cytometry Society (ICCS) [34,35], according to which the PNH clone is evaluated across multiple cell lineages (erythrocytes, neutrophils, monocytes). Evaluating erythrocytes alone may distort the true size of the PNH clone due to prior blood transfusions and/or complement-mediated lysis of defective red blood cells. Determining GPI-deficient neutrophils and monocytes is a more accurate test for assessing the true size of the PNH clone [30,36]. High-sensitivity flow cytometry can detect PNH clones of 0.01% and larger.

2.4 Instrumental Diagnostics

Instrumental examinations in PNH are used to diagnose potential disease complications and identify comorbid conditions that may affect the course and prognosis of the underlying disease.

- complex ultrasound examination of the abdominal organs;

- color Doppler ultrasound of the lower extremity veins;

GCP CLEL 5Grade of Recommendation – C (Level of Evidence – 5)

- echocardiography [26];

GIB CLOE 4Grade of Recommendation – C (Level of Evidence – 4)

Comments: when characteristic complications such as thrombosis in various locations are identified in patients with PNH, additional investigations are performed (as clinically indicated), including complex abdominal ultrasound, contrast-enhanced magnetic resonance angiography, mesenteric vessel Doppler ultrasound, and duplex scanning of the inferior vena cava and portal system to confirm thrombosis and determine its exact location [13,38]. The use of intravenous contrast must be strictly justified due to the additional risk of nephropathy and acute kidney injury associated with active intravascular hemolysis.

2.5 Other Procedures

Comments: bone marrow evaluation should include morphological, cytochemical (sideroblast staining), karyological, and, where possible, molecular analysis of myelokaryocytes to differentiate classic PNH from forms associated with another hematological disorder (aplastic anemia, myelodysplastic syndrome, primary myelofibrosis).

When aplastic anemia or MDS is suspected, adult patients should undergo bone marrow histopathology and FISH testing in accordance with guidelines for the respective disorders.

A spontaneous decrease in PNH clone size may indicate clonal evolution to MDS or AML and warrants repeat bone marrow examination.

3. Treatment, including drug and non-drug therapies, diet therapy, and pain management

Currently, disease-modifying therapy for PNH involves agents that suppress complement system activity and thereby prevent complement-dependent intravascular lysis of erythrocytes. Three drugs are approved in the Russian Federation for the targeted therapy of PNH patients: eculizumab**, ravulizumab, and pegcetacoplan.

Eculizumab** is a humanized monoclonal antibody against the C5 complement component administered intravenously.

Ravulizumab is a long-acting humanized monoclonal antibody against the C5 complement component designed for intravenous administration (with an dosing interval of 56 ± 7 days).

Pegcetacoplan is administered subcutaneously and is a small molecule of 40 kDa consisting of two identical pentadecapeptides covalently linked to a polyethylene glycol (PEG) molecule. The peptide fragments bind to complement C3 and inhibit the activation of the entire complement cascade, leading to the suppression of both the characteristic intravascular hemolysis of PNH and the extravascular hemolysis resulting from opsonization of erythrocytes by C3d complement components.

Ravulizumab and pegcetacoplan were approved by the Russian Ministry of Health in 2023. There is currently no substantial local experience with the use of these medicinal products in PNH patients in our country.

3.1 Pathogenetic therapy

Eculizumab therapy** leads to the cessation of intravascular hemolysis, elimination or reduction of transfusion dependence, a lower frequency of thrombotic complications, improvement or stabilization of renal function, decreased severity of pulmonary hypertension, and a significant increase in the life expectancy of patients with PNH [25,26,29,40–43].

A history of thrombotic complications in the development of which intravascular hemolysis plays a leading role

Transfusion dependence due to chronic hemolysis

Presence of high disease activity, including symptoms of smooth muscle dystonia caused by hemolysis (dysphagia, abdominal pain, chest pain, erectile dysfunction, etc.)

Impairment of renal functional capacity due to intravascular hemolysis, including episodes of acute kidney injury and criteria for chronic kidney disease.

GCR ALEL 2Grade of Recommendation – A (Level of Evidence – 2)

Comment: eculizumab therapy** is associated with an increased risk of infectious complications, primarily meningococcal infection (Neisseria meningitidis). Accordingly, vaccination with a meningococcal vaccine (group J07AH Vandals/Vaccines for meningitis prevention according to the ATC classification) is indicated for patients at least 2 weeks before starting eculizumab therapy**, followed by revaccination in accordance with standards existing in the Russian Federation. In emergency cases where eculizumab therapy** must be started earlier than 2 weeks after vaccination, the patient should be prescribed prophylactic antibiotic therapy [47].

If pregnancy occurs in a PNH patient who does not have the above indications, the decision to initiate and continue pathogenetic therapy with eculizumab** is made by a medical board involving hematologists who are experts in the treatment of PNH.

If breakthrough intravascular hemolysis develops during regular eculizumab therapy**, its association with potential complement-activating factors and the interval since the previous drug administration should be assessed. In cases of typical recurrent breakthrough hemolysis 1–4 days before the next eculizumab administration**, adjustment of the eculizumab dosing regimen may be required**: #increasing the dose to 1200 mg every 14 days or shortening the interval between administrations of the standard 900 mg dose to 12 days. An alternative tactic is switching the patient to ravulizumab therapy [37,46,48]. Additional indications for switching to ravulizumab therapy, considering its prolonged duration of action, are pediatric age and the absence of or extremely difficult venous access in patients. Ravulizumab therapy, similarly to eculizumab, requires up-to-date vaccination with a meningococcal vaccine (group J07AH according to the ATC classification).

In a number of PNH patients, despite regular eculizumab therapy** and the absence of laboratory signs of intravascular hemolysis, anemia persists with a requirement for red blood cell suspension replacement transfusions, which should be regarded as a suboptimal response to pathogenetic therapy. Criteria for optimal and suboptimal treatment response are presented in Table 1.

The cause of persistent transfusion-dependent anemia during eculizumab therapy** is the activation of extravascular (synonym: intracellular) hemolysis, the pathogenesis of which is associated with the activation of proximal complement and opsonization of PNH erythrocytes by C3d complement components followed by phagocytosis and destruction of erythrocytes by the macrophage system. Laboratory criteria for extravascular hemolysis include a positive direct Coombs test, reticulocytosis >120 thousand/µl, hyperbilirubinemia due to the indirect fraction >1.5xULN in the absence of reliable laboratory markers of intravascular hemolysis (LDH >1.5xULN, presence of hemosiderin in urine, elevated plasma free hemoglobin). The presence of pronounced extravascular hemolysis in patients receiving eculizumab for a prolonged period (more than 6 months) may serve as an indication for switching to pegcetacoplan therapy. The administration of pegcetacoplan, which blocks the entire complement cascade, requires vaccination against three pathogens: Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. The subsequent revaccination schedule is determined by the instructions for use of the administered vaccines. In the absence of vaccination against one or more pathogens, prophylactic antibiotic therapy is required [49].

Another cause of a suboptimal hematological response to eculizumab** is bone marrow failure. Accordingly, in patients with 2–3 lineage cytopenia, a bone marrow examination is necessary to rule out the development of bone marrow aplasia [50].

The decision to switch a patient from eculizumab therapy** to ravulizumab or pegcetacoplan is made by a medical board involving hematologists who are experts in the field of PNH treatment.

Pathogenetic drug therapy can be carried out in a 24-hour hospital setting, as well as at the maintenance therapy stage – in a day hospital or outpatient setting.

Table 1. Criteria for the efficacy of pathogenetic therapy for PNH with complement inhibitors [50]:

If a suboptimal hematological response persists for 3 months or more (provided the pathogenetic therapy regimen is followed), it is necessary to investigate the causes of insufficient treatment efficacy and, if possible, take appropriate corrective measures.

3.2 Hematopoietic stem cell transplantation

Hematopoietic stem cell transplantation (HSCT – A16.05.001 Bone marrow transplantation) is currently the only radical method of treating PNH; however, performing HSCT in adult patients is associated with a high incidence of severe complications and, accordingly, high mortality. In this regard, the 'classic' form of PNH is no longer an indication for HSCT provided that pathogenetic treatment is available.

Comments: in PNH associated with other hematological diseases (AA, MDS), HSCT may be considered as a therapeutic option according to the indications accepted for these diseases [51–53].

Comments: eculizumab therapy** is administered before transplantation and for 4 weeks after transplantation.

Comments: the risk of allogeneic HSCT is increased in patients with a history of thrombosis. The decision regarding transplantation in such patients must be made collectively, involving expert specialists from federal centers.

3.3 Supportive care

Patients with a mild course of PNH in the absence of indications to start pathogenetic therapy are monitored by a local hematologist with regular monitoring of clinical status and key laboratory parameters reflecting hemolysis activity, degree of anemia, functional capacity of the liver and kidneys, coagulation status, and iron metabolism.

Comments: When performing blood transfusions in patients with PNH, it is necessary to be guided by the transfusion medicine standards and guidelines currently in force in the Russian Federation. The previously existing theory that only washed erythrocytes should be used as a transfusion medium has not been confirmed in clinical studies [55]. Various red blood cell-containing blood components may be used for transfusion; however, it should be remembered that patients with marked transfusion dependence may develop alloimmunization over time, and therefore leukoreduced blood components should be preferred. It is also important to bear in mind the potential development of post-transfusion iron overload in patients receiving regular transfusions of red blood cell-containing blood components.

When assessing indications for replacement blood transfusions, a combination of factors must be taken into account: the patient's age, the presence of concomitant and intercurrent diseases, hypoxia tolerance, and social factors (ability to work, profession, etc.).

Comments: Recurrent thrombosis is a frequent complication of PNH. In the event of acute thrombosis of any localization, hospital admission and immediate initiation of anticoagulant therapy are required. Low-molecular-weight heparins (ATC heparin group – B01AB: dalteparin sodium, enoxaparin sodium**, nadroparin calcium, parnaparin sodium**, bemiparin sodium) at therapeutic doses are preferred. The decision on the duration of anticoagulant therapy, including prophylactic therapy after the resolution of thrombosis, should be made individually, taking into account concomitant diseases, the degree of thrombocytopenia, and the availability of disease-modifying therapy, since standard regimens for prophylactic anticoagulation have not yet been developed. As a rule, thrombotic complications are rare during treatment with eculizumab**. Data on the use of direct factor Xa inhibitors (ATC group B01AF) in patients with PNH are currently limited to isolated reports.

Comments: The prescription of glucocorticoids and iron preparations in patients with PNH is associated with an increased incidence of severe complications: infections, thrombosis, renal failure, and increased transfusion dependence.

Comments: Folic acid and vitamin B12 deficiency frequently develops in patients with PNH, requiring additional metabolic therapy to correct the deficiency.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

There are no special rehabilitation methods for patients with PNH. The need for rehabilitation may arise when life-threatening complications develop, such as cerebral venous thrombosis or avascular necrosis of the femoral heads complicated by secondary coxarthrosis. In such cases, indications for neurorehabilitation or arthroplasty followed by orthopedic rehabilitation are determined in the manner established for the respective conditions.

5. Prevention and Dispensary Observation

There is no specific prevention for PNH.

Patients with PNH require ongoing follow-up by a hematologist and, if necessary, by specialists in other fields depending on the nature and severity of the developed complications (e.g., in case of extrahepatic portal hypertension due to portal system thrombosis, consultation and follow-up by a portal surgeon are required).

The frequency of follow-up examinations is determined individually (from 3 to 12 months) and depends on the severity of the PNH course, the frequency of hemolytic crises, the nature of treatment (disease-modifying therapy or replacement blood transfusions), the presence and nature of complications, and concomitant diseases.

Monitoring of patients with PNH includes tracking routine parameters as well as monitoring the size of the PNH clone.

- for a baseline PNH clone >1% – every 6 months, as well as immediately in case of clinical deterioration or the development of thrombotic complications [30,32,56];

- for a baseline PNH clone <1% – every 12 months [30,32,56].

GCR CLEL 3Grade of Recommendation — C (Level of Evidence — 3)

- complete blood count (CBC) with differential;

- blood reticulocyte count;

- general clinical blood chemistry panel (determination of alanine aminotransferase, aspartate aminotransferase, LDH, creatinine, urea, conjugated bilirubin, unconjugated bilirubin);

GCP CLEL 5Grade of Recommendation – C (Level of Evidence – 5)

- Direct antiglobulin test (direct Coombs test);

- General clinical blood chemistry panel (determination of ferritin, iron-binding capacity of serum, iron);

- Determination of vitamin B12 (cyanocobalamin) blood levels;

- Assessment of serum folic acid levels;

- Echocardiography (if there is a history of pulmonary hypertension or concomitant cardiac pathology).

GCP CLEL 5Grade of Recommendation – C (Level of Evidence – 5)

6. Organization of Medical Care

Indications for emergency hospital admission of patients with PNH:

1. Development of a hemolytic crisis and/or thrombotic complications and/or a severe infectious-inflammatory process (pneumonia, sepsis, especially in patients receiving disease-modifying therapy).

Indications for planned hospitalization in a medical organization:

1. Dispensary examination, including at the time of initial diagnosis;

2. Dispensary examination to monitor the efficacy of disease-modifying therapy;

3. Persistence or increase in transfusion dependence, relapses of breakthrough hemolysis in patients regularly receiving disease-modifying therapy.

Indications for patient discharge from a medical organization

1. Stabilization of clinical and hematological parameters during disease-modifying or symptomatic therapy;

2. Resolution of infectious complications.

Conditions for administering disease-modifying therapy (inpatient/outpatient):

Given its good tolerability, disease-modifying therapy with intravenous complement inhibitors can be administered in a 24-hour inpatient setting, a day care setting, or on an outpatient basis. Subcutaneous medications may be administered independently by the patient after appropriate training and upon satisfactory tolerability.

7. Additional Information

Additional factors are not presented in these guidelines

Criteria for Evaluating the Quality of Medical Care

References

Appendix A1. Working Group Composition

Members of the National Hematological Society:

• Parovichnikova E.N.¹, MD, DSc, General Director of the National Medical Research Center for Hematology, Chief freelance hematologist of the Russian Ministry of Health

• Lukina E.A.¹, MD, DSc, Professor, Head of the Department of Orphan Diseases, National Medical Research Center for Hematology

• Mikhailova E.A.¹, MD, DSc, Professor, Leading Researcher of the Sector for Research in Hemoblastosis Chemotherapy, Hematopoiesis Depressions, and Bone Marrow Transplantation

• Latyshev V.D.¹, Head of the Hematology Day Hospital for Patients with Hereditary Metabolic Diseases, National Medical Research Center for Hematology

• Tsvetayeva N.V.¹, PhD, Senior Researcher, Sector for the Study of Non-Malignant Blood System Diseases

• Ponomarev R.V.¹, PhD, Head of the Sector for the Study of Non-Malignant Blood System Diseases, National Medical Research Center for Hematology.

• Fidarova Z.T.¹, PhD, Head of the Department of Hemoblastosis Chemotherapy and Hematopoiesis Depressions with a Bone Marrow and Hematopoietic Stem Cell Transplantation Unit, National Medical Research Center for Hematology

• Galtseva I.V.¹, MD, DSc, Head of the Blood and Bone Marrow Cell Immunophenotyping Sector, National Medical Research Center for Hematology

• Dvirnyk V.N.¹, PhD, Head of the Centralized Clinical Diagnostic Laboratory, National Medical Research Center for Hematology

• Ptushkin V.V.², MD, DSc, Chief freelance hematologist of the Moscow Healthcare Department, Deputy Chief Physician for Hematology at the S.P. Botkin City Clinical Hospital of the Moscow Healthcare Department, Professor of the Department of Oncology, Hematology, and Radiology, Pirogov Russian National Research Medical University of the Ministry of Health of Russia

• Kulagin A.D.³, MD, DSc, Director of the R.M. Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantology, Pavlov First Saint Petersburg State Medical University of the Ministry of Health of Russia, Chief freelance hematologist of the Northwestern Federal District

• Shilova E.R.⁴, PhD, Hematologist, Russian Research Institute of Hematology and Transfusiology of the Federal Medical-Biological Agency

• Mitina T.A.⁷, MD, DSc, Prof., Head of the Department of Clinical Hematology and Immunotherapy, M.F. Vladimirsky Moscow Regional Research Clinical Institute (MONIKI), Chief freelance hematologist of the Ministry of Health of the Moscow Region

Members of the Russian Society of Pediatric Oncologists and Hematologists:

• Maschan A.A.⁵, MD, DSc, Professor, Corresponding Member of the RAS, Deputy General Director, Director of the Institute of Hematology, Immunology, and Cell Technologies, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Ministry of Health of Russia

• Smetanina N.S.⁵, MD, DSc, Professor, Deputy Director of the Institute of Hematology, Immunology, and Cell Technologies, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Ministry of Health of Russia

Members of the Federation of Laboratory Medicine:

• Lugovskaya S.A.⁶, MD, DSc, Professor of the Department of Clinical Laboratory Diagnostics, Russian Medical Academy of Continuous Professional Education (RMANPO) of the Ministry of Health of Russia

1. National Medical Research Center for Hematology of the Ministry of Health of the Russian Federation

2. S.P. Botkin City Clinical Hospital of the Moscow Healthcare Department

3. R.M. Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantology, Pavlov First Saint Petersburg State Medical University of the Ministry of Health of Russia

4. Russian Research Institute of Hematology and Transfusiology of the Federal Medical-Biological Agency

5. Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology (Dmitry Rogachev NMRCPHOI) of the Ministry of Health of the Russian Federation

6. Russian Medical Academy of Continuous Professional Education (RMANPO) of the Ministry of Health of the Russian Federation

7. M.F. Vladimirsky Moscow Regional Research Clinical Institute (MONIKI)

Conflict of interest: the authors declare no conflict of interest.

Appendix A2. Methodology for Developing Clinical Guidelines

Target audience for these clinical guidelines:

1. Hematology specialists

2. General practitioners / Internal medicine specialists

3. Neurology specialists

4. Nephrology specialists

5. Pulmonology specialists

Methodology for evidence collection

• Search for publications in specialized periodical print media

• Search in electronic databases.

Databases used for evidence collection/selection:

The evidence base for the guidelines includes publications indexed in the Cochrane Library, PUBMED, and MEDLINE databases. The search depth was 30 years.

Methods used for assessing the quality and strength of evidence:

• Expert consensus;

• The significance of evidence was assessed in accordance with the grading scale of levels of evidence certainty (LEC) for diagnostic methods (Table 1) and for prevention, treatment, and rehabilitation methods (Table 2). The significance of the recommendations was assessed in accordance with the scale for grading the strength of recommendations (Table 3)

Table 1. Levels of Evidence (LoE) grading scale for diagnostic methods (diagnostic interventions)

Table 2. Scale for grading levels of evidence certainty (LEC) for prevention, treatment, and rehabilitation methods (preventive, therapeutic, and rehabilitative interventions)

Table 3. Scale for grading grades of recommendation (GR) for prevention, diagnostic, treatment, and rehabilitation methods (preventive, diagnostic, therapeutic, and rehabilitative interventions)

Procedure for updating clinical guidelines.

The mechanism for updating clinical guidelines provides for their systematic revision at least once every three years, as well as upon the emergence of new evidence-based medicine data on the diagnosis, treatment, prevention, and rehabilitation of specific diseases, or in the presence of justified additions/comments to previously approved clinical guidelines, but no more frequently than once every 6 months.

Appendix A3. Reference Materials

Appendix A3.1. Hematopoietic stem cell transplantation in patients with paroxysmal nocturnal hemoglobinuria aged 0 to 18 years.

1. General provisions

Hematopoietic stem cell transplantation (HSCT) – a medical technology used in the treatment of malignant blood diseases that are incurable by other (conservative) methods. The principle of the method consists of a combined effect on the hematopoietic and lymphoid systems affected by the disease, which comprises 1) the immuno- and myelosuppressive effect of high-dose chemo- and radiation therapy; 2) replacement of bone marrow hematopoietic function with donor hematopoietic stem cells; and 3) an immunological (alloimmune) effect exerted by donor immunocompetent cells on the recipient's residual lymphohematopoiesis.

2. HSCT stages and clinical periodization

The HSCT procedure consists of the following stages (periods):

1. Pre-transplant evaluation of the donor and recipient

2. Pre-transplant conditioning of the donor and recipient

3. Recipient conditioning

4. Graft harvesting and processing

5. Myeloinfusion (transplantation proper)

6. Immunosuppressive and supportive therapy until myeloid reconstitution (graft engraftment)

7. Immunosuppressive and supportive therapy at the early post-engraftment stage (days 30–100)

8. Immunosuppressive and supportive therapy at the late post-engraftment stage (days 100–365)

1. Selection of Donor and HSC Source [59]

To ensure timely donor selection, all patients with PNH who have indications for HSCT undergo human leukocyte antigen (HLA) typing of the patient and potential related donors. Family member typing tactics are determined by the clinic's strategy regarding the use of HSC donors. The donor for a patient with PNH may be (in order of decreasing priority):

a. Fully matched related donor

b. Partially matched related (haploidentical) donor, or matched unrelated donor, or partially matched unrelated donor with an acceptable degree of mismatch

c. Unrelated cord blood

The source of HSCs is chosen based on the HSCT technological platform implemented at the clinic. The source of HSCs may be:

• Bone marrow (BM)

• Peripheral blood stem cells (PBSC)

• Cord blood (CB)

2. Pre-HSCT Patient Evaluation [60]

Pre-HSCT patient evaluation aims to establish the status of the underlying disease, assess comorbidity, functional status, nutritional status, infectious status, and psychological status.

Clinical laboratory diagnostic methods used in pre-HSCT patient evaluation [60]:

• Complete blood count (CBC) with differential

• General clinical blood chemistry test including the following parameters: albumin, total protein, total bilirubin, direct bilirubin, creatinine, urea, glucose, electrolytes (K, Na, Mg, Ca), alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, lactate dehydrogenase, alpha-amylase, pancreatic amylase, total cholesterol, triglycerides, lipase, low-density lipoproteins, high-density lipoproteins

• Assay of blood ferritin level, serum folic acid level, and vitamin B12 (cyanocobalamin) level in blood

• Blood gas and acid-base balance analysis

• Peripheral blood immunophenotyping to identify lymphocyte subpopulation composition (main subsets)

• Blood immunoglobulin level assay

• Qualitative PCR detection of Epstein-Barr virus DNA in peripheral and cord blood

• Qualitative PCR detection of Cytomegalovirus DNA in peripheral and cord blood

• Qualitative PCR detection of human herpesvirus type 6 (HHV-6) DNA in peripheral and cord blood

• PCR detection of human adenovirus DNA in nasopharyngeal/oropharyngeal swabs

• Microbiological (culture) blood test for sterility

• Determination of ABO blood groups and Rh factor (D antigen)

• Indirect antiglobulin test (Coombs test), detection of antibodies to erythrocyte antigens in blood, detection of antibodies to blood group antigens, detection of cold anti-erythrocyte antibodies in blood

• Direct antiglobulin test (direct Coombs test)

• Determination of prothrombin time in blood or plasma, and international normalized ratio (INR)

• Determination of activated partial thromboplastin time

• Determination of thrombin time in blood

• Detection of antibodies to Aspergillus spp. in blood

• Hospital screening: detection of hepatitis B virus surface antigen (HBsAg) in blood, detection of antibodies to hepatitis B virus core antigen in blood, molecular biological testing of blood for hepatitis C virus, molecular biological testing of blood for Treponema pallidum, detection of IgM and IgG antibodies to human immunodeficiency virus HIV-1 in blood, detection of IgM and IgG antibodies to human immunodeficiency virus HIV-2 in blood

• Quantitative PCR detection of hepatitis B virus DNA in blood

• Quantitative PCR detection of hepatitis C virus RNA in blood

• Rapid immunochromatographic stool test for Clostridium difficile toxins A and B

• Microbiological testing for major causative agents of infectious diseases from mucous membranes/skin surface

• General clinical cerebrospinal fluid (CSF) analysis: physical properties of CSF, microscopic examination of CSF, cell counting in a counting chamber (determination of pleocytosis), cytological examination of CSF cells

• Urinalysis, microscopic examination of urinary sediment

• Protein determination in urine

• Urine glucose assay

• Urine creatinine assay

• Urine urea assay

• Microbiological (culture) testing for sterility of blood, cerebrospinal fluid, urine, and fragments of medical devices (central venous catheters, implants)

• Microbiological testing for major causative agents of infectious diseases in body fluids (exudates, transudates)

• Microbiological testing for major causative agents of infectious diseases in biopsy specimens

• Microbiological testing for major causative agents of infectious diseases in bronchoalveolar lavage/sputum

• Microbiological testing for major causative agents of infectious diseases in wound discharge

• Microbiological testing for major causative agents of infectious diseases in feces/rectal swab

• Urine calcium assay

• Urine phosphorus assay

• Urine potassium, sodium, calcium, and chloride assay

• Pancreatic amylase activity assay in blood

• Blood troponin I and T assay

• Assessment of nephron function via creatinine clearance (Rehberg's test)

• Cell line sorting by flow cytometry (flow cytometric analysis of biological material)

• Fluorescence in situ hybridization (FISH) (after hypotonic cell treatment, on prepared cell suspension, imprints, and smears) (1 DNA probe)* (molecular cytogenetic study (FISH method) for one chromosome pair)

• Differential leukocyte count (leukogram)

• Free triiodothyronine (fT3) blood test

• Free thyroxine (FT4) blood test

• Thyroid-stimulating hormone (TSH) blood test

• Total cortisol blood test

• Insulin-like growth factor I blood test

• Blood serum follicle-stimulating hormone assay, blood serum luteinizing hormone assay

• Blood total estradiol assay, blood total testosterone assay

• Determination of class M and G antibodies (IgM, IgG) to Cytomegalovirus in blood

• Determination of class G antibodies (IgG) to Epstein-Barr virus capsid antigen (VCA) in blood

• Determination of class M antibodies (IgM) to Epstein-Barr virus capsid antigen (VCA) in blood

• Determination of class G antibodies (IgG) to Varicella-Zoster virus in blood

• Determination of class M antibodies (IgM) to Varicella-Zoster virus in blood

• Determination of class G antibodies (IgG) to Herpes simplex virus types 1 and 2 in blood

• Determination of class M antibodies (IgM) to Herpes simplex virus types 1 and 2 in blood

• Determination of class G antibodies (IgG) to Toxoplasma gondii in blood

• Determination of class M antibodies (IgG) to Toxoplasma gondii in blood

Instrumental methods used in patient evaluation prior to HSCT:

• Targeted chest X-ray (2 projections)

• Computed tomography of the abdominal cavity and retroperitoneal space with intravenous bolus contrast enhancement

• Spiral computed tomography of the paranasal sinuses

• Computed tomography of the chest

• Brain magnetic resonance imaging with contrast under general anesthesia — 3T (Tesla)

• Ultrasound examination of the kidneys, adrenal glands, urinary bladder, abdominal cavity organs (liver, gallbladder, pancreas, spleen), retroperitoneal space, pelvic organs

• Electrocardiography; recording, analysis, description, and interpretation of electrocardiographic data (children aged 1–18 years)

• 24-hour ambulatory blood pressure monitoring

• Cardiopulmonary exercise testing (Ergospirometry)

• Duplex scanning of brachiocephalic arteries with color Doppler blood flow mapping

• Duplex scanning of the inferior vena cava and portal system veins

• Acquisition of bone marrow cytologic specimen via puncture, bone marrow smear cytologic examination (myelogram)

• Lumbar puncture

• Anesthetic management (including early postoperative care)

• Esophagogastroduodenoscopy

• Colonoscopy

• Endoscopic biopsy of the esophagus, stomach, duodenum

• Endoscopic colon biopsy

• Rectal biopsy using video-endoscopic technologies

• Skin biopsy

• Diagnostic bronchoscopy

• Electroencephalography with video monitoring

• Orthopantomography

Specialist physician consultations in the pre-transplant period:

• Ophthalmologist

• Otolaryngologist

• Neurologist

• Dentist

• Medical psychologist

• Cardiologist

• Pulmonologist

• Endocrinologist

• Obstetrician-gynecologist

3. Donor evaluation prior to HSCT [61]

Donor evaluation prior to HSCT is performed in two stages. Stage 1: evaluation of the potential donor. Aimed at determining the degree of HLA antigen compatibility and identifying absolute contraindications to donation. Stage 2: evaluation of the actual donor. Aimed at an objective assessment of the donor's health status and infection status, identifying relative contraindications to donation, and determining the priority method for HSC collection.

Clinical laboratory diagnostic methods used in donor evaluation prior to HSCT [61]:

• Complete blood count (CBC) with differential

• General clinical chemistry blood test including the assessment of the following parameters: albumin, total protein, total bilirubin, direct bilirubin, creatinine, urea, glucose, electrolytes (K, Na), alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, alpha-amylase, pancreatic amylase, total cholesterol, triglycerides, lipase, low-density lipoproteins, high-density lipoproteins

• Determination of ABO blood groups and Rh factor (D antigen)

• Determination of prothrombin time in blood or plasma, and international normalized ratio (INR)

• Determination of activated partial thromboplastin time

• Determination of thrombin time in blood

• Detection of antibodies to Aspergillus spp. in blood

• Hospital screening: detection of hepatitis B virus surface antigen (HBsAg) in blood, detection of antibodies to hepatitis B virus core antigen in blood, molecular biological testing of blood for hepatitis C virus, molecular biological testing of blood for Treponema pallidum, detection of IgM and IgG antibodies to human immunodeficiency virus HIV-1 in blood, detection of IgM and IgG antibodies to human immunodeficiency virus HIV-2 in blood

• Quantitative PCR detection of hepatitis B virus DNA in blood

• Quantitative PCR detection of hepatitis C virus RNA in blood

• General clinical urinalysis, microscopic examination of urine sediment

• Determination of class M and G antibodies (IgM, IgG) to Cytomegalovirus in blood

• Determination of class G antibodies (IgG) to Epstein-Barr virus capsid antigen (VCA) in blood

• Determination of class M antibodies (IgM) to Epstein-Barr virus capsid antigen (VCA) in blood

• Determination of class G antibodies (IgG) to Varicella-Zoster virus in blood

• Determination of class M antibodies (IgM) to Varicella-Zoster virus in blood

• Determination of class G antibodies (IgG) to Herpes simplex virus types 1 and 2 in blood

• Determination of class M antibodies (IgM) to Herpes simplex virus types 1 and 2 in blood

• Determination of class G antibodies (IgG) to Toxoplasma gondii in blood

• Determination of class M antibodies (IgG) to Toxoplasma gondii in blood

Instrumental methods used in donor evaluation prior to HSCT [61]:

• Targeted chest X-ray (2 projections)

• Ultrasound examination of the kidneys, adrenal glands, urinary bladder, abdominal cavity organs (liver, gallbladder, pancreas, spleen), retroperitoneal space, pelvic organs

• Electrocardiography; recording, analysis, description, and interpretation of electrocardiographic data

• 24-hour ambulatory blood pressure monitoring

• Duplex scanning of brachiocephalic arteries with color Doppler blood flow mapping

• Duplex scanning of the inferior vena cava and portal system veins

• Echocardiography

• Anesthetic management (including early postoperative care)

4. Graft collection [62]

HSC graft collection and processing include procedures for retrieving donor HSCs and their subsequent processing aimed at establishing optimal functional characteristics of the graft. The stages of graft collection are:

• Donor preparation

• Graft collection

• Graft processing

• Graft quality control

• Graft transportation and storage (when the location and time of graft collection differ from those of graft infusion)

Donor preparation

Donor preparation depends on the graft collection method and, accordingly, on the chosen HSC source (BM or PBSC).

When bone marrow is used as the HSC source, donor preparation includes:

• Preoperative evaluation

• Vascular access establishment

When used as a HSC source, donor preparation includes:

• Pharmacological mobilization of HSCs into the bloodstream. The goal of pharmacological mobilization of HSCs is the temporary relocation of bone marrow HSCs into the vascular bed to enable the collection of the required number of HSCs.

• Vascular access establishment

Graft collection

Collection of donor BM [63]

Donor BM collection is performed in the operating room under general or epidural anesthesia. Technically, BM collection consists of successive punctures in the region of the posterior superior iliac spine and iliac crest, followed by BM aspiration. The target volume of donor BM is 20 ml/kg of the recipient's body weight and must not exceed 20 ml/kg of the donor's body weight. The aspirated BM is transferred into a heparinized container (bag).

Medicines and medical supplies required for donor BM collection [64]:

• Gelatin** – 4% solution

• Propofol**

• Rocuronium bromide**

• Sugammadex**

• Sevoflurane**

• Fentanyl**

• Ropivacaine**

• Endotracheal tube

• Peripheral venous catheter

• Bone marrow aspiration needle

• Medical syringe 10 ml, 5 ml, 2 ml capacity

• Bone marrow collection set (or blood component collection set)

• Infusion set

• Sodium heparin**

• Dextrose + sodium citrate

• Suture material

• Scalpel

• Adhesive bandage

• Sterile gloves

• Antiseptics and disinfectants for skin preparation of the surgical field

Collection of donor PBSCs [65]

Donor PBSC collection is performed in the blood transfusion department or by a transfusiologist of the HSCT department. Technically, donor PBSC collection is performed by automated leukapheresis using a blood cell separator. The leukapheresis volume is determined based on the blood HSC concentration resulting from pharmacological mobilization and ranges from 0.5 to 2 circulating blood volumes (CBV).

Medicines and medical supplies required for donor PBSC collection [65]:

• Colony-stimulating factors (ATC group L03AA)

• #Plerixafor 0.24 mg/kg/day s.c.

• Fistula needles 17G

• Peripheral catheter 16G

• Central venous catheter 7-12F

• Spectra Optia Collection Set (ref 10110, Terumo BCT) or Spectra Optia IDL Set (ref 10310, Terumo BCT)

• Dextrose + sodium citrate

• Packed red blood cells

• Blood transfusion sets

• Discofix (three-way stopcock)

• Empty sterile blood component bag

Cord blood collection [66]

Cord blood collection is performed in the delivery room. Technically, cord blood collection is carried out by puncturing the umbilical vein of the umbilical cord and collecting the cord blood into a collection system (four-chamber bag with anticoagulant).

HSC graft processing [67]

Graft processing is a set of technical procedures aimed at altering the qualitative and quantitative parameters of the HSC graft to ensure optimal functional characteristics in terms of the efficacy and safety of the HSCT procedure. Graft processing is performed in a specialized laboratory within the blood transfusion department or HSCT department. The main types of graft processing are [68]:

• Graft splitting

• Graft volume reduction

• Plasma reduction (removal)

• Erythrocyte reduction (removal)

• HSC selection (CD34 selection)

• Selective lymphocyte depletion (ab T depletion, CD19 depletion, CD45RA depletion)

• Cryopreservation and thawing

The choice of processing method (or combination of methods) is determined by the baseline characteristics of the donor-recipient pair, the HSC source, and the technological platform implemented in the transplant center.

Graft quality control

An HSC graft is a suspension of donor nucleated cells enriched with HSCs. Prior to administering the HSC graft to the recipient, quality control of the graft is mandatory.

The mandatory HSC graft parameters subject to control and registration are:

• Graft volume

• Concentration and absolute count of nucleated cells

• Concentration and absolute count of CD34+ cells (HSC-enriched fraction)

Additional HSC graft parameters subject to control and registration are

• Concentration and absolute count of leukocyte subpopulations (depending on the chosen graft processing method)

• CD34+ cell viability (HSC-enriched fraction)

Transportation and storage of the HSC graft

Transportation and storage of the HSC graft (or a portion of it) are necessary when graft collection, processing, and infusion are separated in time and space. HSC graft transportation can be performed in the native state or after cryopreservation. Native graft transportation is carried out at a temperature range of +3 to +8 ^°C, maintaining the cold chain. The maximum transportation and storage time (including processing) without cryopreservation is 72 hours from the moment of HSC graft collection. Cryopreserved graft transportation is carried out at a temperature range of -150 to -196 ^°C, maintaining the cold chain.

5. Conditioning regimen [69]

The conditioning regimen is a high-dose chemotherapy +/- radiotherapy program aimed at eradicating the tumor (leukemia) and the recipient's hematopoietic and immune systems, thereby creating the conditions necessary for the engraftment and function of donor hematopoiesis, the development of the donor immune system, and the graft-versus-leukemia (GVL) effect. Conditioning regimens include chemotherapeutic agents with pronounced immunosuppressive and/or myelosuppressive effects. The choice of drugs, doses, and sequence of administration within the conditioning regimen may vary. As a rule, conditioning regimens utilize high doses of chemotherapy associated with a high risk of dose-dependent side effects. The choice of conditioning regimen is determined by the HSCT technological platform implemented at the transplant center and the baseline characteristics of the donor-recipient pair.

Medicaments used in various conditioning regimens in PNH (total cumulative dose) [69]:

• #Treosulfan, 30-42 g/m² [70]

• #Busulfan**, 8 mg/kg, oral formulation [71]

• #Fludarabine**, 100-180 mg/m² [72,73]

• Thiotepa, 3.38-8.10 mg/kg

• #Cyclophosphamide**, 40-200 mg/kg [73]

• Total body irradiation, 2-6 Gy

6. Introduction to GvHD [74]

Graft-versus-host disease (GvHD) is a key immunological complication of HSCT. GvHD is an immunopathological process based on the recognition by donor immunocompetent cells of antigenic differences between the donor and recipient, the generation of an immune response, and inflammatory damage to the recipient's organs and tissues. There are 4 categories of GvHD: 1) classic acute GvHD (aGvHD); 2) late acute GvHD; 3) 'overlap syndrome' of acute and chronic GvHD; 4) chronic GvHD (cGvHD). According to the NIH Consensus, the latter two categories form the broad category of chronic GvHD.

1) Classic aGvHD: a clinical syndrome developing within up to 100 days after HSCT or donor lymphocyte infusion (DLI). The basis – acute inflammatory organ damage and dysfunction. Classic target organs: skin, gastrointestinal tract (GIT), liver. Skin involvement manifests as maculopapular rash, epidermolysis. GIT involvement – diarrhea, nausea, vomiting, hemorrhagic colitis, ileus. Liver involvement – hepatitis with a predominance of cholestasis.

2) Late aGvHD: typical clinical manifestations of aGvHD developing after day 100 post-HSCT or donor lymphocyte infusion (DLI). Frequently observed upon discontinuation of immunosuppressive therapy.

3) 'Overlap syndrome' of aGvHD and cGvHD: a combination of typical clinical manifestations of both aGvHD and cGvHD, regardless of the time of onset.

4) cGvHD: chronic immune-mediated damage to organs and tissues based on impaired development of immunological tolerance. Histopathologically characterized by progression to fibrosis.

The main risk factors for the development of GvHD include: HLA-mismatch between donor and recipient (in the classic approach to GvHD prophylaxis), female donor for a male recipient, use of PBSCs as a graft source, donor alloimmunization (history of pregnancies, blood transfusions), prior treatment history of the patient, myeloablative conditioning, presence of cytomegalovirus infection.

7. GvHD prophylaxis [75]

The goal of GvHD prophylaxis is to prevent the development of GvHD overall, and particularly severe, life-threatening, and disabling forms of GvHD. GvHD prophylaxis can be based on the targeted depletion of GvHD effectors from the HSC graft ex vivo (see Graft Collection section) and on pharmacological intervention in the recipient. The list of drugs used in GvHD prophylaxis is presented below [76]. Drugs are grouped into GvHD prophylaxis regimens; the choice of regimen is determined by the risk of GvHD in the donor-recipient pair and the HSCT technological platform implemented at the transplant center.

• #Cyclosporine**, 3 mg/kg/day

• #Methotrexate**, 10-15 mg/m²/day

• #Tacrolimus**, 0.02-0.03 mg/kg/day [77]

• #Mycophenolate mofetil**, 30 mg/kg/day

• #Cyclophosphamide**, 50 mg/kg/day [78]

• #Antithymocyte globulin**, rabbit 1.5-2.5 mg/kg/day, total dose 2.5-10 mg/kg [79]

• #Bortezomib**, 1.3 mg/m²/day [80]

• #Abatacept**, 10 mg/kg (no more than 800 mg) every 2 weeks for the first 3 doses, then every 4 weeks [81]

• #Tocilizumab**, 4-8 mg/kg once a week [82]

• #Sirolimus, 4-12 mg/day [83]

• #Everolimus**, 1.5 mg/m² twice daily [84]

• #Rituximab**, 375 mg/m²/week [85]

3. Graft engraftment (myeloreconstitution) [4]

The first stage in evaluating HSCT efficacy is achieving graft engraftment. Graft engraftment criteria include: achieving a leukocyte count >1,000/µL, neutrophil count >500/µL, and platelet count >20,000/µL for 3 consecutive days. An additional criterion for engraftment is the presence of donor chimerism in the bone marrow. Primary graft failure can be diagnosed if engraftment criteria are not met within 30 days; however, using certain transplantation technologies (e.g., post-transplant cyclophosphamide** or double umbilical cord blood transplantation), engraftment may occur at a later date. Opportunistic viral infections may also delay graft engraftment.

8. Diagnosis and treatment of acute GvHD [86,87]

The diagnosis of aGvHD is based on the observation and recording of characteristic clinical manifestations and symptoms in the recipient following HSC graft infusion. To verify the clinical diagnosis of aGvHD, a biopsy of the involved organ and histological examination must be performed. The severity of aGvHD is assessed according to the international grading system shown in Tables 1 and 2 [86,87].

Once a clinical diagnosis of aGVHD is established, therapy is initiated; its regimen is determined by the severity of aGVHD. The severity of aGVHD and the response to first-line therapy determine the prognosis of aGVHD course and survival. In the absence of a response to first-line therapy, a decision is made to prescribe second- and subsequent-line therapy.

Clinical laboratory diagnostic methods used in the diagnosis and treatment of GVHD [86,87]:

• Complete blood count (CBC) with differential

• General clinical blood chemistry analysis including the following parameters: albumin, total protein, total bilirubin, direct bilirubin, creatinine, urea, glucose, electrolytes (K, Na, Mg, Ca), alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase, alpha-amylase, pancreatic amylase, total cholesterol, triglycerides, lipase, low-density lipoproteins, high-density lipoproteins

• Blood gas and acid-base balance analysis

• Peripheral blood immunophenotyping to identify lymphocyte subpopulation composition (main subsets)

• Blood immunoglobulin level assay

• Molecular genetic study of hematopoietic chimerism after unrelated bone marrow transplantation

• Qualitative PCR detection of Epstein-Barr virus DNA in peripheral and cord blood

• Qualitative PCR detection of Cytomegalovirus DNA in peripheral and cord blood

• Qualitative PCR detection of human herpesvirus type 6 (HHV-6) DNA in peripheral and cord blood

• PCR detection of human adenovirus DNA in nasopharyngeal/oropharyngeal swabs

• Microbiological (culture) blood test for sterility

• Determination of ABO blood groups and Rh factor (D antigen)

• Indirect antiglobulin test (Coombs test), detection of antibodies to erythrocyte antigens in blood, detection of antibodies to blood group antigens, detection of cold anti-erythrocyte antibodies in blood

• Direct antiglobulin test (direct Coombs test)

• Individual erythrocyte mass compatibility (Cross-matching test using recipient immune antibodies and donor major histocompatibility complex antigens)

• Determination of prothrombin time in blood or plasma, and international normalized ratio (INR)

• Determination of activated partial thromboplastin time

• Determination of thrombin time in blood

• Detection of antibodies to Aspergillus spp. in blood

• Hospital screening: determination of hepatitis B virus (Hepatitis B virus) surface antigen (HbsAg) in blood, determination of antibodies to hepatitis B virus (Hepatitis B virus) core antigen in blood, molecular biological study of blood for hepatitis C virus (Hepatitis C virus), molecular biological study of blood for Treponema pallidum, determination of immunoglobulin M and G (IgM, IgG) antibodies to human immunodeficiency virus HIV-1 (Human immunodeficiency virus HIV 1) in blood, determination of immunoglobulin M and G (IgM, IgG) antibodies to human immunodeficiency virus HIV-2 (Human immunodeficiency virus HIV 2) in blood

• Quantitative PCR detection of hepatitis B virus DNA in blood

• Quantitative PCR detection of hepatitis C virus RNA in blood

• Rapid immunochromatographic stool test for Clostridium difficile toxins A and B

• Microbiological testing for major causative agents of infectious diseases from mucous membranes/skin surface

• General clinical cerebrospinal fluid (CSF) analysis: physical properties of CSF, microscopic examination of CSF, cell counting in a counting chamber (determination of pleocytosis), cytological examination of CSF cells

• Urinalysis, microscopic examination of urinary sediment

• Protein determination in urine

• Urine glucose assay

• Urine creatinine assay

• Urine urea assay

• Microbiological (culture) testing for sterility of blood, cerebrospinal fluid, urine, and fragments of medical devices (central venous catheters, implants)

• Microbiological testing for major causative agents of infectious diseases in body fluids (exudates, transudates)

• Microbiological testing for major causative agents of infectious diseases in biopsy specimens

• Microbiological testing for major causative agents of infectious diseases in bronchoalveolar lavage/sputum

• Microbiological testing for major causative agents of infectious diseases in wound discharge

• Microbiological testing for major causative agents of infectious diseases in feces/rectal swab

• Urine calcium assay

• Urine phosphorus assay

• Urine potassium, sodium, calcium, and chloride assay

• Pancreatic amylase activity assay in blood

• Blood troponin I and T assay

• Assessment of nephron function via creatinine clearance (Rehberg's test)

• Cell line sorting by flow cytometry (flow cytometric analysis of biological material)

• Fluorescence in situ hybridization (FISH) (after hypotonic cell treatment, on prepared cell suspension, imprints, and smears) (1 DNA probe)* (molecular cytogenetic study (FISH method) for one chromosome pair)

Instrumental methods and procedures used in the diagnosis and treatment of GVHD [86,87]:

• Targeted chest X-ray (2 projections)

• Computed tomography of the abdominal cavity and retroperitoneal space with intravenous bolus contrast enhancement

• Spiral computed tomography of the paranasal sinuses

• Computed tomography of the chest

• Brain magnetic resonance imaging with contrast enhancement under anesthesia – 3T (Tesla)

• Ultrasound examination of the kidneys, adrenal glands, urinary bladder, abdominal cavity organs (liver, gallbladder, pancreas, spleen), retroperitoneal space, pelvic organs

• Electrocardiography recording; interpretation, description, and analysis of electrocardiographic data (children aged 1–18 years)

• 24-hour ambulatory blood pressure monitoring

• Cardiopulmonary exercise testing (Ergospirometry)

• Diffusing capacity of the lungs test (children >10 years)

• Duplex scanning of brachiocephalic arteries with color Doppler blood flow mapping

• Serial echocardiography (provided to inpatients)

• Duplex scanning of the inferior vena cava and portal system veins

• Acquisition of bone marrow cytologic specimen via puncture, bone marrow smear cytologic examination (myelogram)

• Lumbar puncture

• Anesthetic management (including early postoperative care)

• Esophagogastroduodenoscopy

• Colonoscopy

• Endoscopic biopsy of the esophagus, stomach, duodenum

• Endoscopic colon biopsy

• Rectal biopsy using video-endoscopic technologies

• Skin biopsy

• Diagnostic bronchoscopy

• Electroencephalography with video monitoring

Medications used in the treatment of GVHD (daily dose) [87–89]:

• #Methylprednisolone**, 2 mg/kg/day

• Rabbit antithymocyte globulin** — rabbit 1.5–2.5 mg/kg/day, cumulative dose 2.5–10 mg/kg [79]

• Cyclosporine**, 1–3 mg/kg/day

• #Methotrexate**, 5–15 mg/m²/day [90]

• #Tacrolimus**, 0.03 mg/kg/day [77]

• #Mycophenolate mofetil**, 190–1600 mg/m²/day [91]

• #Cyclophosphamide**, 50 mg/kg/day [78]

• #Alemtuzumab**, 0.3–2 mg/kg (cumulative dose administered over 2–6 days) [92]

• Ibrutinib**, 140–420 mg/mg/day

• #Ruxolitinib**, 2.5–10 mg/m² twice daily [93]

• #Etanercept**, 0.4 mg/kg twice weekly [94]

• #Bortezomib**, 1.3 mg/kg/day [80]

• #Abatacept**, 10 mg/kg (no more than 800 mg) every 2 weeks for the first 3 doses, then every 4 weeks [81]

• #Tocilizumab**, 4-8 mg/kg once a week [82]

• #Sirolimus 2–4 mg/day [83]

• #Everolimus**, 1.5 mg/m² twice daily [84]

• #Infliximab**, 10 mg/kg once weekly [95]

• #Rituximab**, 375 mg/m²/week [85]

• Topical glucocorticoids

• Topical calcineurin inhibitors (from the group "Preparations for the treatment of dermatitis, excluding corticosteroids", D11AH according to the ATC classification) — #tacrolimus**, #pimecrolimus** in external dosage forms [96,97]

• Mesenchymal stromal cells

• #Imatinib**, 65–260 mg/m²/day [98]

9. Diagnosis and treatment of cGVHD [99]

A diagnosis of cGVHD is established when at least one diagnostic manifestation or one distinctive manifestation is identified, confirmed by biopsy, laboratory tests, or imaging studies (Table 3).

The severity of cGVHD is determined based on the number of affected organs and the degree of functional impairment in the affected organ (see Table 4) [101].

• Mild cGVHD – involvement of 1–2 organs or sites (excluding the lungs), without clinically significant functional impairment (maximum score of 1 in all affected organs)

• Moderate cGVHD – involvement of at least one organ or site with clinically significant, but not extensive dysfunction (maximum score of 2), or 3 or more organs without clinical functional impairment (maximum score of 1 in each organ), or lung involvement of no more than 1 point.

• Severe cGVHD – significant dysfunction (score of 3 in any organ), or lung involvement (score of 2 or higher).

Notes.

* ECOG (Eastern Cooperative Oncology Group) scale (see Appendix G1)

** ALP may be elevated in growing children, which does not reflect hepatic dysfunction

*** Assessment of pulmonary function on the scale should be carried out taking into account clinical presentation and pulmonary function tests. Lung Function Score (LFS) is the most common scale reflecting the global assessment of lung function after the diagnosis of bronchiolitis obliterans is established. FEV1 and DLCO percentages must be converted into a numerical scale: >80% = 1; 70–79% = 2; 60–69% = 3; 50–59% = 4; 40–49% = 5; <40% = 6. LFS = FEV1 + DLCO, normal is 2–12.

Abbreviations: KPS – Karnofsky Performance Status, LPS – Lansky Play-Performance Scale, BSA – body surface area, ADL – activities of daily living, GI – gastrointestinal tract, ALP – alkaline phosphatase, ALT – alanine aminotransferase, AST – aspartate aminotransferase, LFS – lung function score, FEV1 – forced expiratory volume in 1 second, DLCO – diffusing capacity of the lungs for carbon monoxide, ROM – range of motion, P-ROM – photographic range of motion

To ensure timely diagnosis and early initiation of therapy, prevent life-threatening conditions and disability, a systematic and thorough assessment of patients' organs and systems is required. This also helps assess response to therapy and determine further treatment strategy. The frequency of systematic assessment of organ involvement and severity of cGVHD is once every 4 weeks.

cGVHD therapy is based on pharmacological immunosuppression. The goal of therapy is to restore the function of affected organs, prevent or minimize disability, and restore quality of life. The baseline drug is prednisolone at a dose of 1 mg/kg/day (or methylprednisolone at an equipotent dose). The approach to additional drug therapy and second-line therapy is determined by the severity of the process, the set of involved organs, and the baseline immunosuppressive therapy at the time of cGVHD manifestation.

10. Prophylaxis and treatment of infectious complications [102]

Secondary immunodeficiency in HSCT recipients develops as a result of the underlying disease, baseline therapy, conditioning regimen, GVHD prophylaxis and treatment regimen, and GVHD itself. The immunodeficiency is combined in nature and includes impairments of all major components of immune defense: 1) disruption of the barrier function of the skin and mucous membranes; 2) disruption of the normal microbiota with loss of colonization resistance; 3) granulocytopenia; 4) monocytopenia; 5) humoral immunodeficiency; 6) cellular immunodeficiency. The nature of immunodeficiency and its severity vary at different stages of HSCT. In the first 30 days (prior to engraftment and in the early post-engraftment period), granulocytopenia and impaired barrier function of the skin and mucous membranes dominate. Between days 30 and 100, cellular immunodeficiency dominates. After 100 days, humoral immunodeficiency persists, while the severity of cellular immunodeficiency varies depending on the volume of immunosuppressive therapy. The nature of immunodeficiency determines the predisposition to developing a specific type of infection and dictates the set of measures for the prophylaxis and treatment of infectious complications in accordance with the post-HSCT period.

a. Bacterial infections [103]

The main risk factors for bacterial infections in HSCT recipients in the early periods are granulocytopenia, the use of central venous catheters, mucositis, dermatitis, preexisting colonization with multidrug-resistant pathogens, and refractory underlying disease. Bacterial infections in the early periods are caused by Gram-positive cocci and Gram-negative rods. In later periods, the main risk factors are humoral immune deficiency and hyposplenism, which predispose to infections by encapsulated microorganisms (pneumococcus, Haemophilus influenzae, meningococcus). The main types of bacterial infections after HSCT are pneumonia, sinusitis, proctitis, soft tissue infections, and bacteremia/sepsis.

Prophylaxis of bacterial infections includes sanitary and epidemiological measures: access control, hand hygiene of staff and caring family members, microbiological safety of food and water. Pharmacological prophylaxis of bacterial infections is possible in accordance with the hospital's infection control policy.

Diagnosis of bacterial infections includes clinical, instrumental, and laboratory diagnostics.

Treatment of bacterial infections in HSCT recipients is carried out on the principle of empirical therapy followed by adjustment according to the results of microbiological testing and phased escalation or de-escalation depending on the dynamics of the infectious process.

b. Viral infections [104]

The main risk factors for viral infections in HSCT recipients are lymphopenia, cellular immunodeficiency, ongoing GVHD, systemic corticosteroid therapy, lymphodepleting monoclonal antibody therapy, the use of unrelated and mismatched donors, the presence of a persistent viral infection in the patient and/or donor prior to HSCT, ex vivo T-cell depletion, and refractory underlying disease. The main viral pathogens in HSCT recipients are cytomegalovirus (CMV), Epstein-Barr virus, human herpesvirus type 6, herpes simplex virus, varicella-zoster virus, parvovirus B19, adenovirus, and respiratory syncytial virus. Viral infections in HSCT recipients may present typically, but a severe course and generalization of the infection are characteristic.

Prophylaxis of viral infections includes sanitary and epidemiological measures: access control, hand hygiene of staff and caring family members, microbiological safety of food and water, microbiological safety of blood components. A specific preventive measure is the selection of a donor matched to the patient regarding exposure to key viral pathogens, and the choice of an HSCT technology that minimizes the volume and duration of post-HSCT pharmacological immunosuppression. Pharmacological prophylaxis of viral infections is carried out against herpes family viruses in accordance with the individual risk of infection using the corresponding group representative.

Diagnosis of viral infections includes clinical, instrumental, and laboratory diagnostics.

Treatment of viral infections in HSCT recipients is carried out on the principle of preemptive therapy and empirical therapy followed by adjustment according to the results of microbiological testing and phased escalation or de-escalation depending on the dynamics of the infectious process. Preemptive therapy is the standard for controlling CMV infection and includes weekly quantitative monitoring of viral load in the patient's blood and the administration of antiviral therapy when threshold values are exceeded. Similar tactics are applied for EBV, adenovirus, and HHV-6 in the high-risk group.

c. Fungal infections [105,106]

The main risk factors for viral infections in HSCT recipients are granulocytopenia, lymphopenia, cellular immunodeficiency, ongoing GvHD, systemic corticosteroid therapy, lymphodepleting monoclonal antibody therapy, use of unrelated and partially matched donors, colonization of mucous membranes with yeasts, invasive fungal infection in the patient prior to HSCT, ex vivo T-cell depletion, and refractory underlying disease. The main fungal pathogens in HSCT recipients are fungi of the genus Candida, Aspergillus, and Zygomycetes. Fungal infections in HSCT recipients may present as superficial mycosis, but a severe course, invasive growth, and generalization of the infection are characteristic.

Prophylaxis of fungal infections includes sanitary and epidemiological measures: air quality control, access control, hand hygiene for staff and caregivers, microbiological safety of food and water. A specific preventive measure is the choice of HSCT technology that minimizes the volume and duration of pharmacological immunosuppression after HSCT. Pharmacological prophylaxis of fungal infections is carried out in accordance with the individual risk of infection in the specific post-HSCT period.

Diagnosis of fungal infections includes clinical, instrumental, and laboratory diagnostics. Depending on the local epidemiology of fungal infections, monitoring of fungal biomarkers and a preemptive treatment strategy may be used.

Therapy for fungal infections in HSCT recipients is carried out on the principle of preemptive therapy and empirical therapy, followed by correction based on the results of microbiological studies and staged escalation or de-escalation depending on the dynamics of the infectious process.

4. Prophylaxis and therapy of specific (organ-specific) complications

d. Hemorrhagic cystitis [107]

Hemorrhagic cystitis is a specific HSCT complication developing due to damage to the bladder mucosa by drugs used in the conditioning regimen and/or an infectious process. The highest risk of developing early hemorrhagic cystitis is characteristic of conditioning regimens that include high doses of cyclophosphamide**, busulfan**, and total body irradiation. Late hemorrhagic cystitis is associated with BK polyomavirus infection, certain adenovirus serotypes, and, less commonly, cytomegalovirus. GvHD plays a certain role in the development of hemorrhagic cystitis. Clinically, hemorrhagic cystitis manifests as dysuria, hematuria (ranging from microhematuria to massive hemorrhage with urethral obstruction by clots). Prophylaxis of hemorrhagic cystitis includes selecting a conditioning regimen that minimizes exposure to high doses of cyclophosphamide**, hyperinfusion regimen during cyclophosphamide** therapy, and systemic administration of mesna**. Therapy for early hemorrhagic cystitis includes infusion, forced diuresis, systemic hemostatic therapy, and local hemostasis. Therapy for late hemorrhagic cystitis includes infusion, forced diuresis, systemic hemostatic therapy, local hemostasis, and antiviral therapy.

e. Hepatic veno-occlusive disease (sinusoidal obstruction syndrome) [108,109]

Hepatic veno-occlusive disease is a specific HSCT complication caused by damage to the endothelium of the portal system sinusoids, development of microthrombosis and occlusion of portal vessels, resulting in portal hypertension and liver failure. Risk factors for hepatic veno-occlusive disease (VOD) include myeloablative conditioning regimens using drugs such as busulfan**, cyclophosphamide**, or etoposide**, as well as total body irradiation. The probability of developing this condition is also increased by fludarabine**, sirolimus, and calcineurin inhibitors. In addition, risks for VOD development include pre-existing liver dysfunction (cirrhosis, fibrosis), hepatitis C, and hepatic iron overload (serum ferritin >1000 ng/dL).

The diagnosis of VOD is established based on the following criteria (presence of two or more symptoms):

• Thrombocytopenia refractory to platelet transfusions

• Unexplained weight gain within 3 days despite diuretic therapy, or weight gain >5% from baseline

• Hepatomegaly

• Ascites

Increase in bilirubin level above baseline within 3 days or ≥2 mg/dL within 72 hours

VOD prophylaxis includes selecting a conditioning regimen in accordance with VOD risk factor analysis, in particular avoiding high doses of busulfan** and combinations of alkylating agents in patients with pre-existing liver damage. Medications used in VOD prophylaxis and therapy are summarized below.

f. Mucositis [110]

Damage to the mucous membrane of the gastrointestinal tract caused by the cytotoxicity of chemotherapy drugs and radiation therapy included in the conditioning regimen. The main risk factors are the composition of the conditioning regimen and the doses of antineoplastic agents. The localization of the lesion varies from localized forms (stomatitis, esophagitis, gastritis, enterocolitis) to generalized damage. A certain contribution to the development of mucositis is made by infections, in particular herpes simplex virus and Candida species. Clinical manifestations of mucositis include pain syndrome, edema, hyperemia and ulceration of the mucosa, dysphagia, and diarrhea syndrome. Mucositis prophylaxis includes oral hygiene, dental care at the patient preparation stage for HSCT, pharmacological suppression of gastric secretion, and antacids. Mucositis therapy is symptomatic, with the central task being adequate pain relief and treatment with antiseptic and disinfecting agents.

g. Posterior reversible encephalopathy syndrome (PRES) [111]

Posterior reversible encephalopathy syndrome (PRES). A specific neurological complication of HSCT based on local subcortical reversible vasogenic edema of the brain tissue. It is characteristically associated with calcineurin inhibitor therapy and arterial hypertension. The clinical picture includes headache, seizures, visual disturbances, encephalopathy, and focal neurological deficits. The diagnosis is verified by characteristic changes on brain MRI. Therapy includes withdrawal of drugs associated with PRES development and control of arterial hypertension.

h. Respiratory distress syndrome [112]

Acute respiratory distress syndrome (RDS) is a frequent complication after HSCT and may develop as part of a septic process, engraftment syndrome, cytokine release syndrome, or transfusion-related acute lung injury (TRALI). RDS in HSCT recipients is caused by increased permeability of the pulmonary capillary bed and the development of non-cardiogenic pulmonary edema. Clinically manifested by respiratory failure, hypoxemia, and crepitant rales upon auscultation. Imaging is characterized by diffuse reduction in lung transparency. No specific RDS prophylaxis has been developed. Therapy includes respiratory support, ranging from oxygen supplementation to mechanical ventilation, high doses of corticosteroids, and strict fluid balance management.

i. Thrombotic microangiopathy [113]

HSCT-associated thrombotic microangiopathy (TA-TMA) is a heterogeneous group of disorders characterized by microangiopathic hemolytic anemia and consumption thrombocytopenia due to clot formation in the microvasculature, which causes ischemic organ damage.

During the development of TA-TMA, endothelial injury leads to the activation of inflammatory and procoagulant cascades, ultimately resulting in capillary occlusion. However, unlike other endothelial syndromes, complement dysregulation may play a role in the development of TA-TMA. This in turn promotes the production of the C4d complement fraction and the C5b-9 membrane attack complex. Clinical and laboratory manifestations are presented in Table 7. Risk factors for TA-TMA include the use of calcineurin inhibitors, viral infections (CMV, adenovirus, BK virus, and several others), fungal infections, and GvHD. TA-TMA therapy includes dose reduction or discontinuation of calcineurin inhibitors, symptom-based supportive care, and specific drug therapy (see below).

11. Nutritional support [114]

HSCT recipients face significant nutritional deficiency caused by a combination of factors: stomatitis, esophagitis, gastroenterocolitis, disruption of the gut microbiome, loss of appetite, nausea/vomiting, and thermal processing of food. Correction of nutritional insufficiency is based on clinical nutrition technologies, including enteral and parenteral nutrition. Enteral and parenteral drugs used in the clinical nutrition of HSCT recipients are listed below.

12. Transfusion therapy [115]

During HSCT, the recipient's hematopoiesis is replaced by donor hematopoiesis. HSCT leads to transient hematopoietic aplasia, lasting from several days to several weeks. Hematopoietic aplasia typically develops within the first 2–3 weeks following conditioning, but may also occur at later times post-HSCT as a result of graft dysfunction of various etiologies. The following blood components are used in replacement transfusion therapy:

1. Packed red blood cells

2. Platelet concentrate

3. Granulocyte concentrate

4. Fresh frozen plasma

The donor and recipient may be incompatible across various blood group antigen systems. Depending on the type of ABO incompatibility, major (major), minor (minor), and mixed (major+minor) types are distinguished. Major ABO incompatibility (major) implies the presence of agglutinins in the recipient's plasma that are active against donor red blood cells, for example, when performing HSCT from a donor with blood group A(II) to a patient with blood group O(I). Minor ABO incompatibility (minor) is due to the reverse situation, i.e., the presence of agglutinins in the HSC donor's plasma active against the recipient's red blood cells, for example, during HSCT from a donor with blood group O(I) to a recipient with blood group A(II). Mixed (major+minor) incompatibility occurs when both the donor and the recipient have agglutinins active against the recipient's and donor's red blood cells, respectively. Due to the likelihood of severe complications associated with ABO blood group incompatibility between the HSC donor and recipient, rules for transfusion therapy following transplantation (starting from the day of myeloinfusion) have been formulated, as shown in Table 5.

Table 5. Rules for transfusion therapy in patients after HSCT

Prophylaxis of post-transfusion graft-versus-host disease.

Post-transfusion (blood component transfusion-associated) graft-versus-host disease (PT-GVHD) is one of the most severe and in most cases fatal complications in immunocompromised patients. The pathogenesis of PT-GVHD is based on the engraftment of T-lymphocytes transfused along with blood components, their subsequent activation, proliferation, and cytotoxic damage to various recipient target organs, such as the skin, liver, intestines, and bone marrow. In rare cases, PT-GVHD can sometimes also develop in immunocompetent patients, for example, when the donor and recipient share similar HLA haplotypes.

The standard procedure for PT-GVHD prophylaxis is gamma irradiation of blood components at a dose of 25 Gy [116].

Appendix A3.2. Brief prescribing information for eculizumab**.

International Nonproprietary Name: eculizumab**.

Dosage form: Concentrate for solution for infusion.

Composition: 1.0 ml of concentrate contains the active substance: eculizumab** (rEclz) – 10.0 mg; excipients: sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate heptahydrate, sodium chloride, polysorbate 80, water for injections – up to 1.0 ml.

Indications for use:

Paroxysmal nocturnal hemoglobinuria (PNH). The efficacy of eculizumab** has been confirmed in patients with hemolysis and concomitant clinical symptoms indicative of high disease activity, regardless of historical blood transfusion requirements;

Atypical hemolytic uremic syndrome (aHUS)

Contraindications:

Hypersensitivity to eculizumab**, hamster proteins, or other components of the drug.

Breastfeeding period.

Active Neisseria meningitidis infection.

Lack of vaccination against Neisseria meningitidis (unless appropriate prophylactic antibiotic therapy is administered for 2 weeks following vaccination).

Dosage and administration:

Intravenous infusion over 25–45 minutes for adults and over 1–4 hours for pediatric patients. The treatment course for adult patients (≥18 years) includes a 4-week initial cycle followed by a maintenance therapy cycle.

Initial cycle: 600 mg of eculizumab** once weekly for 4 weeks.

Maintenance therapy: 900 mg of eculizumab** at week 5, followed by 900 mg of eculizumab** every 14±2 days.

For PNH patients under 18 years of age, the dose of eculizumab** is determined based on the child's weight:

Recommendations for solution preparation and infusion administration

Do not mix eculizumab** during intravenous administration with any other drug in the same syringe or vial!

Using a sterile syringe and needle (not supplied), withdraw the entire contents of the eculizumab** vial(s) and transfer the recommended dose into an infusion bag/vial containing one of the following injection solutions: 0.9% sodium chloride solution, 0.15% sodium chloride solution, or 5% dextrose (glucose) solution to obtain an infusion solution with a concentration of 5.0 mg/ml. The volume of the prepared infusion solution containing 5.0 mg/ml of eculizumab** should be: 60 ml (for a 300 mg dose), 120 ml (for a 600 mg dose), 180 ml (for a 900 mg dose), and 240 ml (for a 1200 mg dose).

The prepared infusion solution must be clear and colorless. If the solution is discolored or contains particulate matter, it must not be used.

Immediately prior to administration, gently swirl the infusion vial to homogenize the contents. The temperature of the infusion solution should be 20–25 °C. If the infusion is delayed for any reason, the prepared infusion solution may be stored at 2 to 8 °C for no more than 24 hours without freezing. After this period (24 hours), any unused solution must be discarded.

Adverse reactions

The most frequent adverse reaction during eculizumab** treatment was headache (noted mainly during the initial treatment cycle). The most serious adverse reaction was meningococcal sepsis.

Appendix A3.3. Brief prescribing information for ravulizumab.

International Nonproprietary Name: ravulizumab.

Pharmaceutical form: Concentrate for solution for infusion (300 mg/3 mL; 1100 mg/11 mL; 300 mg/30 mL)

Composition:

300 mg/3 mL: each mL of concentrate contains 100 mg of ravulizumab. Each vial contains 300 mg of ravulizumab. Excipients with known effect: sodium 4.6 mg.

1100 mg/11 mL: each mL of concentrate contains 100 mg of ravulizumab. Each vial contains 1100 mg of ravulizumab. Excipients with known effect: sodium 16.8 mg.

Indications for PNH:

Ravulizumab is indicated for the treatment of adult and pediatric patients weighing 10 kg or more with PNH:

- patients with hemolysis with clinical symptoms indicative of high disease activity;

- patients whose clinical condition is stable after treatment with eculizumab for at least the preceding 6 months.

Contraindications:

- Hypersensitivity to ravulizumab or to any of the excipients;

- Patients with unresolved Neisseria meningitidis infection at the time

treatment is initiated;

- Patients who are currently not vaccinated against Neisseria meningitidis infection, unless they receive appropriate prophylactic

antibiotics for 2 weeks after vaccination.

Dosage and Administration:

Dosing regimen

Adult patients with PNH:

the recommended dosing regimen consists of a loading dose followed by maintenance doses administered via intravenous infusion. The choice of drug dose depends on the patient's body weight, as shown in Table 1.

In adult patients (≥18 years), maintenance doses should be administered every 8 weeks, with the first maintenance dose administered 2 weeks after the loading dose. The administration schedule allows for rare deviations of ± 7 days from the scheduled infusion date (except for the first maintenance dose of ravulizumab), but the subsequent dose should be administered according to the original schedule. For patients switching from eculizumab therapy to ravulizumab therapy, the ravulizumab loading dose should be administered 2 weeks after the last eculizumab infusion, followed by maintenance doses every 8 weeks. The first maintenance dose is administered 2 weeks after the loading dose, as shown in Table 1.

Table 1. Ravulizumab dosing regimen by body weight in adult patients weighing at least 40 kg.

¹The first maintenance dose of the drug is administered 2 weeks after the loading dose.

Pediatric population

The dosing regimen for pediatric patients with PNH weighing ≥40 kg is the same as for adults (Table 1). The weight-based doses and administration frequency for pediatric patients weighing from ≥10 kg to <40 kg are presented in Table 3. For patients switching from eculizumab therapy to ravulizumab therapy, the ravulizumab loading dose should be administered 2 weeks after the last eculizumab infusion. Maintenance doses should then be administered in accordance with the weight-based dosing regimen, as shown in Table 3. The first maintenance dose is administered 2 weeks after the loading dose.

Table 2. Ravulizumab dosing regimen by body weight for pediatric patients with PNH or aHUS weighing less than 40 kg.

¹The first maintenance dose of the drug is administered 2 weeks after the loading dose.

The use of ravulizumab in pediatric patients with PNH weighing less than 30 kg has not been studied. The dosing regimen of ravulizumab in pediatric patients weighing less than 30 kg is based on the dosing regimen in pediatric patients with aHUS, taking into account pharmacokinetic/pharmacodynamic data in PNH patients treated with ravulizumab.

Method of administration:

For intravenous infusion only.

This medicinal product must be administered through a 0.2 µm filter and must not be administered as an intravenous push or bolus.

Ravulizumab is supplied in 3 mL and 11 mL vials (100 mg/mL) and must be diluted to a final concentration of 50 mg/mL. After dilution, Ravulizumab is administered via intravenous infusion using a syringe pump or infusion pump over a minimum period of 0.17 to 1.3 hours (10 to 75 minutes) depending on body weight.

The prepared solution should be used immediately after dilution. Chemical and physical in-use stability has been demonstrated for up to 24 hours at 2-8 ℃ and up to 4 hours at room temperature.

Undesirable effects

The most common adverse drug reactions are diarrhea, upper respiratory tract infections, nasopharyngitis, and headache. The most serious adverse reactions in clinical trials are meningococcal infection and meningococcal sepsis.

Appendix A3.4. Summary of product characteristics for pegcetacoplan.

INN: Pegcetacoplan.

Pharmaceutical form: Solution for subcutaneous injection.

Indications for use:

Pegcetacoplan is indicated for the treatment of adult patients with paroxysmal nocturnal hemoglobinuria (PNH) who have persistent anemia after treatment with a C5 inhibitor for at least 3 months.

Mechanism of action:

Pegcetacoplan is a symmetrical molecule consisting of two identical pentadecapeptides covalently bound to the ends of a linear 40 kDa PEG molecule. The peptide fragments bind to complement C3 and provide broad inhibition of the complement cascade.

Method of administration:

Subcutaneous. Pegcetacoplan should be administered exclusively as a subcutaneous infusion using a syringe infusion pump. This medicinal product can be self-administered.

Dosing regimen:

Pegcetacoplan can be self-administered by the patient or caregiver after receiving proper training, or administered by a healthcare professional. Pegcetacoplan is administered subcutaneously twice weekly at a dose of 1080 mg using a syringe infusion pump system designed to deliver volumes up to 20 mL. The recommended dose of the drug should be administered twice weekly on Day 1 and Day 4 of each treatment week.

Undesirable effects:

The most common adverse drug reactions (ADRs) in patients treated with Pegcetacoplan were injection site reactions: injection site erythema, injection site pruritus, injection site swelling, injection site pain, and injection site bruising.

Appendix B. Physician Action Algorithms

Appendix C. Patient Information

What is paroxysmal nocturnal hemoglobinuria (PNH)?

PNH is a rare acquired hematological disorder. PNH primarily affects the bone marrow, where all blood cells are produced. Due to a spontaneous acquired mutation in one or more hematopoietic stem cells, defective red blood cells are produced that become vulnerable to the body's own immune system (PNH clone). These defective red blood cells undergo destruction within blood vessels under the action of immune surveillance proteins. The process of red blood cell destruction is called "hemolysis". As a result of hemolysis, a large amount of hemoglobin is released from the destroyed red blood cells; when excreted in the urine, it causes the urine color to turn dark or even black with a reddish tint (hemoglobinuria).

Who gets paroxysmal nocturnal hemoglobinuria?

The disease can affect people regardless of gender, ethnicity, or age, although the peak incidence occurs between 30 and 40 years of age. The disease is extremely rare, affecting only 1-2 people per million per year. In some cases, PNH develops before, after, or against the background of other hematological disorders such as aplastic anemia (AA) and myelodysplastic syndrome (MDS); such cases are referred to as PNH associated with AA or MDS. If other hematological disorders are absent, it is referred to as "classical" PNH. Currently, there are no preventive measures for the development of PNH.

What are the most common symptoms of paroxysmal nocturnal hemoglobinuria?

Symptoms caused by anemia (low blood hemoglobin levels) – you may experience weakness, drowsiness, fatigue, shortness of breath upon mild exertion, and palpitations.

Hemoglobinuria is the excretion of dark, up to black, urine, caused by the presence of a large amount of hemoglobin in the urine.

Jaundice is a yellowish discoloration of the skin and sclera.

Dysphagia (swallowing disorder) – you may experience difficulty swallowing food or liquids, which most commonly occurs during periods of increased hemolysis.

Erectile dysfunction – some patients may experience impaired erection.

Abdominal pain – can occur at any time, but typically worsens during a hemolytic crisis. It is important to remember that abdominal pain may be a consequence of abdominal vessel thrombosis, which requires immediate medical attention.

It is important to understand that the disease progresses differently in different patients; therefore, you may experience all of the above symptoms or none at all.

What factors trigger an increase in hemolysis?

Patients with PNH experience chronic hemolysis – a certain number of red blood cells are constantly destroyed within the vascular bed. However, there are situations when hemolysis significantly increases, leading to a sharp drop in hemoglobin, darkening of the urine, and other aforementioned symptoms (hemolytic crisis). The causes of hemolytic crises can include infections, exposure to extreme temperatures, air travel, and other stressors.

What are the possible complications of PNH?

One of the complications of PNH is vascular thrombosis. Thrombosis can occur in both arteries and veins. If you have a history of thrombotic complications, your doctor may prescribe antithrombotic agents (drugs that reduce blood clotting).

Chronic kidney disease may develop due to the deposition of large amounts of iron in the kidneys against the background of chronic hemolysis.

Bone marrow dysfunction. PNH is always accompanied by varying degrees of bone marrow failure. Bone marrow failure manifests as a decrease in peripheral blood levels of hemoglobin, leukocytes, and platelets (cytopenia). A decrease in the leukocyte count can lead to an increased risk of infectious complications. A decrease in the platelet count can lead to an increased risk of bleeding.

How is PNH diagnosed?

To confirm the diagnosis, it is sufficient to analyze venous blood using a specialized technique (flow cytometry). The test will show the percentage of defective cells (the size of the PNH clone) in your blood. It is important to understand that the size of the PNH clone does not always correlate with the clinical picture; thus, PNH with a 50% clone may present with numerous complications, whereas the disease with a 99% clone may have a relatively benign course.

What treatments exist for PNH?

Currently, three drugs are approved in Russia for the treatment of PNH: eculizumab**, ravulizumab, and pegcetacoplan. Eculizumab is an antibody that blocks complement (an immune system factor that destroys red blood cells). Eculizumab** blocks the hemolysis of defective erythrocytes, but does not affect the production of defective cells in any way. Thus, eculizumab only blocks the final link in the chain of pathological events in PNH, but does not cure the disease. Indications for initiating therapy are determined by a medical panel of hematologists. If there are indications for treatment, eculizumab** therapy should be administered indefinitely, since discontinuation of the drug carries a high risk of hemolysis relapse. The drug is administered intravenously every 14±2 days.

Ravulizumab is a long-acting humanized monoclonal antibody against the C5 complement component designed for intravenous administration (with an dosing interval of 56 ± 7 days).

Pegcetacoplan is intended for subcutaneous administration and is a small molecule that inhibits C3 activity.

Supportive care may include prescribing folic acid, cyanocobalamin**, prophylactic anticoagulant therapy, as well as blood transfusions according to indications determined by your attending physician.

Can I pass the disease on to my children?

Since PNH is an acquired and not a congenital disease, you cannot pass the disease on to your children.

What is the prognosis of the disease in PNH?

In the "classical" form and with effective treatment, life expectancy is comparable to that of the general population. In PNH associated with AA or MDS, the prognosis is somewhat worse and is determined primarily by the treatment options for the underlying hematological disease.

Appendix G. Assessment Scales, Questionnaires, and Other Evaluation Tools

Appendix G1. Karnofsky Performance Scale

Russian title: Karnofsky Performance Scale.

Original title: Karnofsky Performance Status.

Original publication:

Karnofsky DA B.J.H. The clinical evaluation of chemotherapeutic agents in cancer. Evaluation of Chemotherapeutic Agents. // Columbia Univ. Press. 1949. P. 191–205.

Type: rating scale.

Purpose: to assess the patient's level of functioning in terms of their ability to care for themselves, daily activity, and physical capability (walking, working, etc.).

Content:

Note. The key (interpretation) is provided within the scale itself.

Appendix G2. P-ROM Scale

Russian title: Photographic Range of Motion scale

Original title: The Photographic Range of Motion (P-ROM) scale

Source (official developer website, validation publication): Carpenter PA. How I conduct a comprehensive chronic graft-versus-host disease assessment. Blood. 2011;118:2679–87

Type: rating scale.

Purpose: To assess the degree of joint mobility

Content:

Key (interpretation): the sum of scores for all four joints is evaluated. The lower the total score, the more pronounced the restriction of mobility.

Appendix G3. WHO/ECOG performance status scale

Russian title: WHO/ECOG performance status scale.

Original title: The Eastern Cooperative Oncology Group/World Health Organization Performance Status (ECOG/WHO PS).

Source (official developer website, validation publication):

1. https://ecog-acrin.org/resources/ecog-performance-status.

2. Oken M.M., Creech R.H., Davis T.E. Toxicology and response criteria of the Eastern Cooperative Oncology Group // Am. J. Clin. Oncol. Cancer Clin. Trials. 1982. Vol. 5, № 6. P. 649–655.

Type: rating scale.

Purpose: to describe the patient's level of functioning in terms of their ability to care for themselves, daily activities, and physical abilities (walking, work, etc.).

Content:

Note. The key (interpretation) is provided within the scale itself.

Appendix G4. Lung function score

Russian title: Lung Function Score

Original title: The Lung function score (LFS)

Source (official developer website, validation publication): Pavletic SZ, Martin P, Lee SJ, Mitchell S, Jacobsohn D, Cowen EW, et al. Measuring therapeutic response in chronic graft-versus-host disease: National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: IV. Response Criteria Working Group report. Biol Blood Marrow Transplant. 2006;12:252–66. doi: 10.1016/j.bbmt.2006.01.008

Type: rating scale.

Purpose: to evaluate pulmonary functional capacity

Content:

Key (interpretation):

Appendix G2. Lansky performance scale

Russian title: Lansky scale.

Original title: Lansky Performance Status.

Original publication:

Lansky S.B., List M.A., Lansky L.L., Ritter-Sterr C., Miller D.R. The measurement of performance in childhood cancer patients. // Cancer. 1987. Vol. 60, №7. P. 1651-1656.

Type: rating scale.

Purpose: to describe the functional level of a patient in terms of daily activity and physical abilities based on play/activity — for patients under 16 years of age.

Content:

Note. The key (interpretation) is provided within the scale itself.

Appendix G3. WHO/ECOG performance status scale

Russian title: WHO/ECOG performance status scale.

Original title: The Eastern Cooperative Oncology Group/World Health Organization Performance Status (ECOG/WHO PS).

Source (official developer website, validation publication):

1. https://ecog-acrin.org/resources/ecog-performance-status.

2. Oken M.M., Creech R.H., Davis T.E. Toxicology and response criteria of the Eastern Cooperative Oncology Group // Am. J. Clin. Oncol. Cancer Clin. Trials. 1982. Vol. 5, № 6. P. 649–655.

Type: rating scale.

Purpose: to describe the patient's level of functioning in terms of their ability to care for themselves, daily activities, and physical abilities (walking, work, etc.).

Content:

Note. Key (interpretation) is given within the scale itself