Aplastic anemia: clinical guidelines

List of Abbreviations

AA — aplastic anemia

allo-HSCT – allogeneic hematopoietic stem cell transplantation

ATG** – antithymocyte globulin**

G-CSF – granulocyte colony-stimulating factors (L03AA according to the ATC classification)

GPI – glycosylphosphatidylinositol

IST – immunosuppressive therapy

IFN – interferon

BM – bone marrow

MDS — myelodysplastic syndrome

PNH — paroxysmal nocturnal hemoglobinuria

HSC – hematopoietic stem cell

CsA** – cyclosporine **

Terms and Definitions

Acquired aplastic anemia (AA) is a hematological disorder characterized by pancytopenia and a sharp decrease in bone marrow cellularity with a quantitative deficiency of hematopoietic stem cells and committed progenitors caused by cellular autoimmune mechanisms.

Combined immunosuppressive therapy (IST) in patients with AA. The optimal non-transplant therapeutic approach for AA, including a course of antithymocyte globulin** (ATG**) and long-term therapy with cyclosporine** (CsA**).

Programmed treatment of patients with AA is a set of therapeutic measures performed in stages starting from the moment of disease diagnosis, implemented according to a specific algorithm, including hematopoietic cell transplantation from an HLA-genoidentical donor or IST with ATG** and CsA**, and if necessary, repeated courses of ATG** and other treatment modalities (hematopoietic cell transplantation from alternative donors and/or the use of the thrombopoietin receptor agonist eltrombopag), enabling long-term patient survival.

Paroxysmal nocturnal hemoglobinuria clone (PNH clone) is a hematopoietic stem cell (HSC) clone with a mutation in the PIG-A gene, leading to impaired synthesis of glycosylphosphatidylinositol (GPI).

Complete remission is practical normalization of hemogram parameters: hemoglobin >100.0 g/L, granulocytes >1.5×10⁹/L, platelets >100.0×10⁹/L, and no need for blood component replacement therapy.

Partial remission: hemoglobin 90–100.0 g/L, granulocytes >0.5–1.5×10⁹/L, platelets 30–100×10⁹/L, and no need for blood component replacement therapy.

Clinical and hematological improvement (minimal hematological response) is improvement in hemogram parameters (hemoglobin 70.0–85 g/L, granulocytes 0.5–1.0×10⁹/L, platelets >20.0×10⁹/L), and disappearance or significant reduction of dependence on blood component transfusions.

Refractory AA is diagnosed in the absence of a response to ongoing combined IST following a second course of ATG** at 6–9 months from the start of the first course.

Neutropenia is a granulocyte count of less than 0.5×10⁹/L, or less than 1.0×10⁹/L with an expected decrease over the next several days [142].

Febrile status is a single elevation of body temperature ≥38.3°C or a body temperature ≥37.8°C persisting for one hour or more.

Hypothermia is a drop in body temperature below 36 °C. This indicator is an important sign of an infectious process, especially in young children and patients receiving glucocorticosteroids [181].

Fever of unknown origin – a condition characterized by fever in the absence of clinical and radiological manifestations of infection and microbiological data.

Bacteremia is the identification of a microorganism from a blood culture taken during a febrile episode that is not a contaminant. False bacteremia includes the identification of so-called contaminants—skin commensals of the hands: coagulase-negative staphylococci, Propionibacterium spp., Micrococcus spp., etc.—in the case of their single identification [115,142,144].

Proven infection is defined by the identification of a microorganism from sterile body sites (blood, cerebrospinal fluid, urine, biopsy material) in the presence of symptoms of an inflammatory response, or the isolation of a pathogen from non-sterile sites (skin, mucous membranes, gastrointestinal tract) with a corresponding local clinical presentation of infection.

Probable infection is defined as infection without the identification of a microorganism from the studied media in the presence of clinical and/or radiological signs of infection with a rapid response to anti-infective therapy.

Febrile neutropenia is a symptom complex combining the development of fever or hypothermia in a patient meeting criteria for neutropenia. It is the most expected infectious complication and develops in the vast majority of patients with AA. In neutropenic patients, due to impaired barrier function of the skin and gastrointestinal mucosa, translocation of endogenous intestinal flora directly into the bloodstream may occur. During an infectious episode, local clinical symptoms may be absent, and the only sign may be febrile fever/hypothermia, lethargy, or altered consciousness [179,180].

1. Brief information on the disease or condition

1.1 Definition of the disease or condition

Aplastic anemia is a hematological disorder characterized by pancytopenia and a sharp decrease in bone marrow cellularity with a quantitative deficiency of hematopoietic stem cells and committed progenitors caused by cellular autoimmune mechanisms.

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

One of the leading mechanisms of hematopoiesis impairment in AA is considered to be immune aggression directed against hematopoietic progenitor cells [1].

Bone marrow failure in AA develops as a result of the suppression of hematopoietic progenitor cell proliferation by activated T-lymphocytes. Activation of T-lymphocytes, expansion of cytotoxic T-clones, and release of mediators of immune suppression of hematopoiesis (interferon gamma [IFNγ], tumor necrosis factor alpha) or factors stimulating T-lymphocyte proliferation and activation (interleukin-2) lead to impaired proliferation and stimulated apoptosis of progenitor cells, resulting in a significant reduction in the hematopoietic cell pool and the development of bone marrow aplasia [1–3].

The main clinical manifestations of the disease are anemia, hemorrhagic syndrome, and severe infectious complications [2].

In addition, the course of AA may be complicated by the development of clonal disorders such as paroxysmal nocturnal hemoglobinuria (PNH), myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The frequency of clonal complications can reach 32% over 10 years [3,4]. The emergence of clonal hematopoiesis can also be detected at earlier stages of AA. This primarily refers to AA occurring with a PNH clone [5,6]. At the same time, the detection of a clone with a deficiency of glycosylphosphatidylinositol (GPI) proteins does not mean the development of PNH as an independent disease with a picture of classical intravascular hemolysis. The size of the PNH clone during the course of AA can vary up to complete disappearance [7]. Evolution into classical PNH, according to various authors, accounts for 11–17% [8–10].

1.3 Epidemiology of the Disease or Condition

According to epidemiological studies, aplastic anemia occurs with varying frequencies in regions such as Europe, North America, and the Far and Middle East; meanwhile, according to the International Agranulocytosis and Aplastic Anemia Study, the prevalence of AA in European countries is 2 cases per 1 million population per year, with this indicator fluctuating depending on the specific country from 0.6 to 3 or more cases per 1 million population per year [11].

1.4 Features of Coding the Disease or Condition

D61.3 – Idiopathic aplastic anemia

D61.8 – Other specified aplastic anemias

D61.9 – Aplastic anemia, unspecified

1.5 Classification of the disease or condition

Acquired AA:

1. Idiopathic AA

2. Secondary AA

3. “Seronegative” hepatitis

4. Immunopathology

5. Pregnancy

6. Radiation

7. Drugs and chemical toxins

8. Viruses

The following criteria for the severity of acquired AA are distinguished:

Very severe aplastic anemia:

Bone marrow cellularity according to trephine biopsy <25% (or cellularity >25% but <50% with a myeloid element content [i.e., excluding lymphocytes and plasma cells] <30%) and 2 or more of the following indicators:

• Neutrophils <0.2×10⁹/L

• Platelets <20×10⁹/L

• Corrected reticulocytosis <1% (less than 40,000/µL)

Severe aplastic anemia

Bone marrow cellularity according to trephine biopsy <25% (or cellularity 25%–50% with a bone marrow myeloid element content [i.e., excluding lymphocytes and plasma cells] <30%) and 2 or more of the following indicators:

• Neutrophils >0.2×10⁹/L, but <0.5×10⁹/L

• Platelets <20×10⁹/L /µL

• Corrected reticulocytosis <1% (or less than 40,000/µL on automated counting)

Non-severe acquired aplastic anemia

All other cases that do not meet the criteria for severe and very severe AA are classified as non-severe (moderately severe) AA.

When determining the severity of AA, data from at least three blood counts taken over a period of at least 2 weeks are taken into account.

1.6 Clinical presentation of the disease or condition

The manifestation of clinical symptoms of AA can be either sudden, with the development of prominent hemorrhagic syndrome and infectious lesions associated with profound neutropenia (stomatitis, sinusitis, pneumonia, enterocolitis), or gradual, presenting with complaints of weakness, fatigue, decreased exercise tolerance forming a typical anemia syndrome, and the development of hemorrhage.

2. Diagnostics, medical indications and contraindications for diagnostic methods

Many recommended methods for diagnosing the disease and associated conditions have a limited evidence base according to the levels of evidence (LE) and grades of recommendation (GR) scales due to the lack of clinical studies devoted to them. Despite this, they are necessary elements of patient evaluation for diagnosis and treatment selection, as more effective and proven methods have not yet been developed.

Criteria for establishing the diagnosis/condition: AA is diagnosed based on the presence of pancytopenia, absence of hepatosplenomegaly, constitutional symptoms, and symptoms characteristic of acute leukemias, hemophagocytic lymphohistiocytosis, or infections presenting with pancytopenia. A mandatory condition for documenting an AA diagnosis is the demonstration of severe bone marrow hypocellularity (less than 25% of the age-adjusted normal value) according to trephine biopsy data, absence of leukemic cells in the bone marrow aspirate, and negative tests for constitutional bone marrow failure syndromes (primarily Fanconi anemia and congenital dyskeratosis).

The diagnosis of AA is established based on clinical manifestations and laboratory examination data.

• Trilineage cytopenia: anemia (hemoglobin <110 g/L), granulocytopenia (granulocytes <2.0×10⁹/L), thrombocytopenia (platelets <100.0×10⁹/L).

• Absence of leukemic cells and solid tumor cells, and absence of megakaryocytes according to bone marrow aspiration data.

• Bone marrow aplasia in the iliac bone biopsy specimen (dominance of fatty bone marrow >75%).

2.1 Complaints and Anamnesis

• In all patients with suspected or confirmed AA, it is recommended to take a medical history and evaluate complaints regarding blood and hematopoietic disorders [12].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: history-taking should identify potential exposure to toxic or medicinal agents or association with hepatitis B and C. A thorough family history is necessary to rule out congenital anomalies, as well as ascertaining the presence of siblings (brothers and/or sisters) to consider the possibility of allogeneic hematopoietic stem cell transplantation (allo-HSCT).

2.2 Physical Examination

Physical examination helps suspect genetic syndromes characterized by aplastic anemia (Fanconi anemia, congenital dyskeratosis), detect changes characteristic of acute leukemias and solid tumors, and assess the severity of hemorrhagic syndrome and infectious lesions, i.e., evaluate the immediate threat to the patient's life.

Particular attention should be paid to:

- Facial structural anomalies (triangular face, small eyes, epicanthus, fine facial features, microcephaly) and limb anomalies (thumb anomalies, thenar shape, polydactyly, syndactyly, clinodactyly)

- Presence of skin pigmentation features (café-au-lait spots, reticular hyperpigmentation, hyperpigmentation)

- Nail dystrophy, especially on the toes

- Hair color and structure (premature graying, thinning, brittleness, fineness)

- Signs of hemorrhagic syndrome (skin, oral mucosa, eye conjunctiva)

- Presence of oral mucosal leukoplakia

- Epiphora

- Liver and spleen size

- Degree of enlargement, number, localization, and characteristics of lymph nodes

- Presence of hypospadias

• In all patients with suspected or confirmed AA, it is recommended to perform a general physical examination at each consultation [12–14].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: the examination should include inspection of the oral cavity (including the oral mucosa and tonsils), measurement of height, body weight, and body temperature, assessment of the skin status and osteoarticular system, detection of signs of hemorrhagic syndrome, cardiac and pulmonary auscultation, palpation of peripheral lymph nodes, thyroid gland, and abdominal organs to assess non-hematological anomalies that may indicate the presence of constitutional AA.

2.3 Laboratory Diagnostics

• In all patients with suspected AA, as well as in all patients with confirmed AA at least 2 times a week until a hematological response is achieved, and subsequently 1 time per month, it is recommended to perform a complete blood count with determination of the absolute reticulocyte count and platelet count to verify the diagnosis and assess disease dynamics [12,15].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: determining the severity of AA requires 3 consecutive blood tests. During treatment monitoring, a complete blood count is performed at least 2–3 times a week until graft engraftment or response to immunosuppressive therapy is achieved, and subsequently 1 time per month [16].

• In all patients with suspected AA, as well as in all patients with confirmed AA 1 time per week until a hematological response is achieved, and subsequently 1 time per month, it is recommended to perform a general clinical biochemical blood test (urea, creatinine, bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), sodium, potassium, calcium, magnesium) to assess the general condition of the patient, organ and system functions, and to monitor complications of immunosuppressive therapy [17].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients with suspected AA, as well as those receiving red blood cell transfusion support, it is recommended to analyze ferrokinetics parameters (study of serum iron level, blood ferritin level) to assess the degree of iron overload [1,18].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients during AA diagnosis, it is recommended to perform a coagulogram (screening coagulation test) (activated partial thromboplastin time (APTT), prothrombin time, fibrinogen) to assess the presence of coagulation disorders in order to determine supportive care tactics [16,17,19].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients with suspected AA or newly diagnosed AA, if not performed previously, it is recommended to determine ABO blood groups; D antigen of the Rh system (Rh factor); C, c, E, e, Cw, K, k antigen phenotype and red blood cell antibodies to select transfusion media and minimize transfusion reactions [16,17,19].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: considering the high frequency of transfusions and developing alloimmunization to donor blood components, red blood cell transfusions must be performed taking into account the erythrocyte phenotype

• In all patients with suspected AA, it is recommended to perform hospital screening: determination of hepatitis B virus surface antigen (HbsAg) in the blood, hepatitis C virus antibodies, Treponema pallidum antibodies in the blood, human immunodeficiency virus HIV 1/2 IgM and IgG antibodies in the blood. If necessary, molecular biological blood testing for human immunodeficiency virus HIV-1 (Human immunodeficiency virus HIV-1) to determine the need for antiretroviral therapy [16,17,19].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients with suspected AA, it is recommended to determine hepatitis B virus DNA, hepatitis C virus RNA, Epstein-Barr virus DNA, cytomegalovirus DNA, human herpesvirus 6 (HHV6) DNA, and parvovirus B19 DNA by PCR in peripheral blood [16,17,19].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients with suspected AA, it is recommended to study the concentration of folic acid and vitamin B12 (cyanocobalamin) in the blood for differential diagnosis with deficiency anemias [15,17,19].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients with suspected AA, it is recommended to study blood immunoglobulin levels and peripheral blood immunophenotyping to identify lymphocyte subpopulations (main ones) for differential diagnosis with primary immunodeficiencies [17,19].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients during AA diagnosis, it is recommended to perform a diepoxybutane test (DEB test) to rule out Fanconi anemia [12,15,20].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients during AA diagnosis, and every 6–12 months from the start of immunosuppressive therapy, it is recommended to determine clones with glycosylphosphatidylinositol (GPI)-anchored protein deficiency (PNH clones) to detect the combination of AA and PNH, analyze PNH clone size over time, and evaluate clonal evolution to PNH [5-10].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients during AA diagnosis, as well as their siblings, it is recommended to perform low-resolution HLA DNA typing for 6 alleles of the A, B, and DRB1 loci with a consultation at a transplantation center to choose the treatment method and search for a potential bone marrow donor [21].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients during AA diagnosis, it is recommended to perform a bone marrow trephine biopsy from the posterior iliac crest with histological examination. Subsequent routine trephine biopsies for monitoring are not indicated; indications are determined individually, primarily when myelodysplastic syndrome with bone marrow fibrosis is suspected [12,15,22,23].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• In all patients during AA diagnosis, as well as every 12–24 months after achieving a hematological response, it is recommended to obtain a bone marrow cytologic preparation by aspiration of the anterior or posterior iliac crests, perform a bone marrow smear cytologic examination (myelogram) to assess the cellular composition of bone marrow hematopoiesis, and perform cytogenetic testing to predict transformation into MDS/hematological malignancy [12,15,22,23].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: in AA, bone marrow aspiration smears are hypocellular, showing relative lymphocytosis and absent megakaryocytes. Sternal puncture in children is prohibited.

• All patients undergoing AA diagnosis are recommended to undergo a standard cytogenetic study (karyotype) of bone marrow cells and FISH testing for monosomy 7 for differential diagnosis with MDS [24–26].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: FISH testing should be performed using probes most typical for MDS (to detect chromosome 7 abnormalities), despite the absence of cytogenetic aberrations or mitoses in standard cytogenetic testing. The detection of clonal aberrations characteristic of MDS at the onset of pancytopenia, in the presence of other diagnostic criteria for severe and very severe AA and in the absence of genetically proven or clinically probable predisposition syndromes to MDS/AML (e.g., SAMD9, SAMD9L, GATA2, SBDS, etc.), does not rule out the diagnosis of acquired AA, may be transient, and is not a contraindication to IST.

• Patients with a family history of hematological disorders (leukemia, thrombocytopenia, aplastic anemia) and/or onset with non-severe, slowly progressive cytopenia are recommended, whenever possible, to undergo molecular genetic testing using high-throughput sequencing with targeted gene panels or Sanger sequencing to identify the most frequent mutations in genes associated with inherited bone marrow failure syndromes (IBMFS) (e.g., Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, etc.) and/or MDS/AML predisposition syndromes (e.g., mutations in GATA2, SAMD9, SAMD9L, RUNX1 and other genes). Molecular genetic testing by other methods (whole-exome sequencing (WES), whole-genome sequencing (WGS)) may be performed for differential diagnosis with IBMFS, MDS/AML predisposition syndromes, and primary immunodeficiencies (PIDs) [20,27,28].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: The decision regarding the need for molecular genetic testing in a patient (and a potential donor in case of planned HSCT), as well as the choice of testing method, depends on the examination results, the nature of cytopenia, the turnaround time for genetic test results, the urgency of starting treatment, and the choice of a donor for transplantation. In the absence of laboratory data and/or a proven genetic defect confirming IBMFS, genetic testing of related donors is not indicated and may lead to an unjustified delay in HSCT. It is important to remember that a negative molecular genetic test result does not rule out IBMFS or PID. Currently, there is insufficient evidence to perform genetic testing for patients without clinical, laboratory, or anamnestic risk factors indicating IBMFS, especially if HSCT from a related donor is not planned. The decision should be made individually, taking into account the expected benefit from verifying the genetic diagnosis of IBMFS in a particular patient and the potential treatment delay associated with the duration of certain molecular genetic tests (WES, WGS), as well as the inability to rule out a constitutional defect based on negative genetic test results.

Comments: microbiological blood testing is performed prior to the administration of systemic antibacterial drugs. Blood collection is performed from a peripherally inserted central catheter (PICC) or, in its absence, from a peripheral vein during a febrile temperature spike, an episode of hypothermia, chills, and/or hemodynamic instability, or the appearance of septicopyemic foci. If the patient has a multi-lumen PICC and symptoms of catheter-related infection, blood samples must be drawn from each lumen. Reliable results can be achieved by properly selecting culture bottles and ensuring the correct volume of blood collected for testing: for children up to 10-12 kg, 1-3 ml in pediatric bottles; for children over three years old, 6-10 ml in aerobic bottles (it is most optimal to use an automated blood culture analyzer). If an anaerobic etiology of the infectious process is possible, use anaerobic bottles; if a fungal infection is suspected, use mycotic bottles. To avoid contamination and subsequent false interpretation, strict adherence to aseptic technique during blood collection and inoculation is necessary. To increase test sensitivity, drawing blood two to three times during the first 24 hours of fever is indicated. After verification of the microorganism, further blood tests are performed 24-48 hours after initiating antibacterial therapy and until the first negative result is obtained. Repeated microbiological blood testing is necessary if febrile neutropenia persists for 72 hours, as well as before escalating antibacterial therapy [115-118]. An automated blood culture analyzer is used for microbiological blood testing.

Comment: Considering the increasing incidence of antibiotic resistance, it is necessary to determine susceptibility, including to reserve-group systemic antibacterial drugs, as well as to identify resistance determinants.

Comments: microbiological tests are performed for precision antibiotic therapy during treatment in the presence of febrile fever and infectious complications.

2.4 Instrumental Diagnostics

• All patients undergoing AA diagnosis are recommended to undergo a chest X-ray in the anteroposterior projection, ultrasound examination of the abdominal organs (liver, spleen, and lymph nodes) and kidneys, and pelvic organs in girls to rule out concomitant pathology [16,17,19].

LeR CLoE 5Level of recommendation evidence C (level of data validity – 5)

• Patients with AA receiving IST who have suspected infectious complications of IST (primarily in the presence of persistent febrile neutropenia for 72–96 hours, respiratory symptoms such as cough, dyspnea, desaturation, or chest pain) are recommended to undergo chest CT to determine infectious lung damage [29].

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

Comments: Chest CT must be performed upon the development of refractory febrile neutropenia even in the absence of any clinical symptoms from the respiratory system. If signs of pneumonia are detected, tracheobronchoscopy and bronchoalveolar lavage (BAL) must be performed. The scope of the study should be maximal and, if necessary, include: molecular biological testing for respiratory syncytial virus, adenovirus, influenza viruses, metapneumovirus, parainfluenza viruses, rhinoviruses, bocavirus, coronaviruses, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Pneumocystis jirovecii, Mycobacterium tuberculosis complex, cytomegalovirus; microscopic examination of bronchoalveolar lavage fluid for fungi (yeast and mold) and Mycobacterium tuberculosis; microbiological (culture) testing for aerobic and facultatively anaerobic microorganisms, microbiological (culture) testing for fungi (yeast and mold), Legionella pneumophila; and determination of fungal metabolites (galactomannan). BAL results will have the highest diagnostic value if performed before the initiation of anti-infective therapy.

It should be remembered that X-ray examination of the lungs in patients with neutropenia has low diagnostic utility and is indicated only for the diagnosis of complications such as pneumo- or hydrothorax, hydropericardium, as well as for checking the position of central venous catheters, pleural drains, or endotracheal tubes [123, 140, 145, 146, 149].

Comment: depending on the clinical situation, a comprehensive ultrasound of internal organs, echocardiography (to rule out endocarditis), and duplex scanning of the upper extremity veins in the projection of the central venous catheter (to rule out thrombotic deposits associated with the central venous catheter) are performed. CT or MRI of the abdominal cavity, retroperitoneal space, and small pelvis with contrast (to rule out lesions of internal organs and intestines), esophagogastroduodenoscopy, colonoscopy (to rule out erosive-ulcerative processes and pathological infiltration)

2.5 Other Procedures

• For all female patients with the onset of menstruation during AA diagnosis, it is recommended to consult an obstetrician-gynecologist to rule out comorbid pathology and address the issue of prescribing hormonal medications to stop the menstrual cycle and prevent uterine bleeding [12, 16, 19].

GRC CLOE 5Grade of Recommendation C (Level of Evidence – 5)

• For patients diagnosed with AA in the presence of hemorrhagic syndrome and/or any visual disturbances, it is recommended to consult an ophthalmologist to rule out intraocular hemorrhage [12, 16, 19].

GRC CLOE 5Grade of Recommendation C (Level of Evidence – 5)

• For patients diagnosed with AA in the presence of infectious, hemorrhagic, and other ENT complications, it is recommended to consult an otorhinolaryngologist to diagnose comorbid pathology [12, 16, 19].

GRC CLOE 5Grade of Recommendation C (Level of Evidence – 5)

Comment: testing of nasopharyngeal and oropharyngeal swabs to detect respiratory viruses by polymerase chain reaction (PCR) or detecting antigens by fluorescent antibody methods. It is advisable during the season of increased ARI incidence or in the presence of respiratory symptoms. Testing for the novel coronavirus infection COVID-19 is mandatory in all cases of febrile neutropenia.

Comment:

Determination of galactomannan in the blood should only be performed in neutropenic patients who are not receiving antifungal prophylaxis active against molds, with a frequency of 2 times a week.

Comment: If bronchoscopy is not feasible, galactomannan determination can be performed in blood serum.

Comment: the combination of febrile neutropenia and focal liver/spleen lesions requires the exclusion of disseminated/invasive candidiasis.

Comment:

Other methods may be used to detect Cryptococcus, including DNA detection and microscopy. Due to the nonspecific clinical and radiological picture, in addition to central nervous system cryptococcal infection, the diagnostic workup must include invasive mycoses, toxoplasmosis, listeriosis, herpes virus infection, and mycobacterioses, including atypical ones.

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

The modern treatment strategy for children with AA includes allogeneic hematopoietic cell transplantation (bone marrow or peripheral blood hematopoietic progenitors mobilized with colony-stimulating factors (L03AA according to the ATC classification, G-CSF)) from an HLA-genoidentical donor or combined IST, which includes two main drugs with pronounced immunosuppressive effects without concomitant myelotoxicity: antithymocyte globulin** (ATG**) and #cyclosporine** (#CsA**) [30–33].

HLA typing of AA patients and siblings should be performed immediately after diagnosis. If a fully matched related donor is available, AA patients should be consulted at a transplant center.

In addition, the pathogenetic therapy of AA patients may include, apart from immunosuppressive drugs (ATG**, #CsA**), medications aimed at activating the proliferation of hematopoietic progenitor cells – granulocyte colony-stimulating factor and the thrombopoietin receptor agonist eltrombopag. Clinical use of eltrombopag** may be accompanied by a mono-, bi-, or trilineage hematological response in patients with refractory AA, and its use in combination therapy programs for AA patients significantly increases the rate of complete response achievement and overall patient survival [34]. The treatment program may include chelating therapy. Replacement transfusion therapy with red blood cell suspension and platelet concentrate is a fundamental component ensuring patient safety until hematopoiesis recovery [15, 16, 35]. Splenectomy is not indicated in children with AA.

Information on performingallo-HSCT in children with AA is presented in Appendix A3.1.

3.1 Allogeneic hematopoietic cell transplantation from an HLA-genoidentical donor

• For all children with AA who have an HLA-genoidentical (matched for 9/10 or 10/10 alleles – A, B, C, DRB1, DQB1) donor, allogeneic hematopoietic cell transplantation is recommended (see Appendix A3.1) [30–33, 36].

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

Comments: transplantation should be performed in a sterile room with HEPA-filtered positive-pressure air. Sterile bed linen must be changed at least once a day or more often if soiled. The patient receives a low-bacterial diet, i.e., thoroughly heat-treated, excluding raw vegetables and fruits, semi-dry smoked products, candies, chips, "live" yogurts and other fermented milk products containing live bacteria, as well as products containing spices and yeast bread.

Bone marrow transplantation is preferred over G-CSF-mobilized peripheral blood hematopoietic progenitors [37]. The use of the latter is possible if the donor/legal representatives refuse and if the recipient's body weight significantly exceeds the donor's body weight.

• All children with AA after allo-HSCT are recommended to undergo a chimerism analysis on days +30, +100, and +360 post-transplantation, following hematopoietic recovery. Additional chimerism studies are necessary in case of graft function deterioration—i.e., recurrent development of anemia and/or granulocytopenia and/or thrombocytopenia not associated with common causes of graft dysfunction (reactivation of latent cytomegalovirus infection and treatment with ganciclovir**) [19,22,36].

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

• Routine use of G-CSF is not recommended for all children with AA in the post-transplantation period [36].

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

Comments: in the event of a bacterial infection uncontrolled by systemic antibacterial drugs, any fungal infection, or in the presence of incompletely resolved infectious complications at the start of the conditioning regimen, G-CSF may be prescribed starting from day +5 post-transplantation at a dose of 5 mcg/kg of body weight daily subcutaneously or intravenously.

• All children with AA following hematopoietic recovery after allo-HSCT are recommended to undergo chimerism analysis on days +30, +100, and +360 post-transplantation. Additional chimerism studies are required if graft function deteriorates [36].

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

3.2 Combined immunosuppressive therapy as the first-line pathogenetic therapy for AA

• All children with AA who do not have an HLA-matched sibling donor or if the donor has contraindications to donation are recommended to receive combined immunosuppressive therapy (IST) with horse ATG** at a dose of 40 mg/kg/day for 4 days [36,38,43,110].

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

• All children with AA undergoing a course of IST are recommended to receive prophylaxis of invasive mycoses with posaconazole** (for children over 13 years of age), voriconazole** (over 2 years of age), itraconazole, or other systemic antifungal drugs Amphotericin B [liposomal] [39].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: administration of systemic antifungal drugs continues until granulocytes rise above 0.5×10⁹/L. Prophylactic administration of co-trimoxazole [Sulfamethoxazole+Trimethoprim]** is not indicated, given the extreme rarity of Pneumocystis pneumonia in IST recipients.

• All children with AA undergoing a course of IST are recommended long-term therapy with #cyclosporine** (#CsA**). #CsA** is prescribed from day 1 of the horse ATG** course. The initial dose of the drug is 5 mg/kg/day, orally, divided into 2 doses. Subsequently, the daily dose is adjusted based on individual pharmacokinetics and individual drug tolerance. The target trough (i.e., pre-dose) concentration of #CsA** in whole blood should be 150-300 ng/mL. In the absolute absence of a hematological response (180-200 days from the start of the first ATG** course; 90 days after the second ATG** course), #CsA** is discontinued prior to hematopoietic cell transplantation. Upon achieving any hematological response, #CsA** is continued for at least 18 months and at least 6 months after reaching the plateau of the hematological response. Upon achieving a stable best response, a total therapy duration of at least 18 months, and no increase in blood counts for 6 months, #CsA** is tapered by 5% of the dose every 2 weeks. Complete withdrawal of the drug takes a total of 40 weeks [19,42,47].

GCR BLEL 2Grade of Recommendations B (Level of Evidence – 2)

Comments: If renal toxicity (elevated urea and creatinine), arterial hypertension uncontrolled by two drugs, or hepatic toxicity develops, #CsA** is stopped completely, regardless of the dose and blood concentration. After complete resolution of toxicity, #CsA** is resumed at a daily dose equal to ½ of the dose at which toxicity developed. If the whole blood #CsA** level rises above 350 ng/mL, the daily dose of #CsA** is reduced to enter the therapeutic window. The average daily dose of #CsA** throughout the treatment course is determined by tolerability and the achieved concentration level. One should not attempt to increase the #CsA** dose to therapeutic levels in case of poor tolerability. If achieving a concentration within the "therapeutic window" is impossible, one should settle for the maximum tolerated dose of the drug.

• Administration of G-CSF is not recommended for all children with AA during the course of IST [36].

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

• Patients who started the ATG** course with an incompletely resolved bacterial or fungal infection or who develop such an infection are recommended to receive G-CSF after completing the ATG** course [19,40].

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

Comments: if granulocytes fail to rise above 0.5×10⁹/L within 42 days, G-CSF is discontinued. If granulocytes rise above 0.5×10⁹/L, the further administration regimen and dosage of G-CSF are selected individually to maintain granulocytes above 0.5×10⁹/L on each day of therapy.

• All children with AA undergoing a course of IST, simultaneously with the start of standard IST (from day 1 of ATG), are recommended to receive eltrombopag** therapy at age-appropriate dosages (see the medicinal product's instructions for use, section "First-line therapy for severe aplastic anemia") for 6 months or more upon achieving a hematological response. The use of eltrombopag** in combination therapy regimens for AA patients significantly increases the rate of achieving partial and complete response [41-43].

GCR ALEL 1Grade of Recommendation A (Level of Evidence – 1)

• Patients with AA and post-transfusional iron overload are recommended to receive iron chelation therapy with deferasirox** (contraindicated in patients under 2 years of age) at an initial dose of 10 mg/kg/day, with subsequent gradual dose escalation up to 30 mg/kg/day in the absence of signs of drug toxicity [44].

LeRR CLoE 4Level of recommendation confidence C (level of evidence – 4)

Comments: iron overload must be confirmed by T2* MRI. When #CsA** and deferasirox** are used concomitantly, there is a high probability of renal dysfunction; therefore, regular monitoring of creatinine and urea levels is required.

3.3 Therapy for refractory forms and relapses of AA

• Patients with refractory AA are recommended to receive therapy with #eltrombopag** at age-appropriate dosages (see the medicinal product's instructions for use, section "First-line therapy for severe aplastic anemia") for 6 months or more upon achieving a hematological response [45,46].

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

Comments: the initiation of #eltrombopag** therapy is possible concomitantly with the start of the second ATG** course, or in the absence of an effect from 2 conducted courses of combined IST with horse ATG** combined with #CsA**, and when allo-HSCT from an alternative donor is not feasible. Eltrombopag therapy is also possible both in the presence of single-lineage cytopenia (thrombocytopenia) and pancytopenia to improve the obtained treatment results.

• Patients with refractory AA are recommended to undergo a repeated course of therapy with horse ATG** in 3-6 months if the 1st course is ineffective [19,36].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: currently, horse ATG** is also the therapy of choice for a repeated course. An alternative is rabbit ATG** at a course dose of 17.5 mg/kg administered over 5 days.

• Patients with confirmed refractoriness to the 1st course of ATG** therapy are recommended to consult at a transplantation center to evaluate the feasibility of allogeneic hematopoietic blood or bone marrow cell transplantation from an unrelated or haploidentical donor [22,36].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

• Patients with "#cyclosporine** dependence" (worsening of cytopenia/relapse during dose tapering or immediately after complete discontinuation) are recommended to resume #CsA** therapy at the previous dose (see section 3.2 "Combined immunosuppressive therapy as the first-line pathogenetic therapy for AA" of these guidelines) even in the absence of transfusion dependence [33,47].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: if there is no effect after 2-3 months, discuss the issue of administering a course of ATG**.

• In patients with AA relapse, it is recommended to administer a repeated course of horse ATG** at the previous dosing regimen [36].

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

• In patients with AA, systemic corticosteroid therapy is not recommended [12,16].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: glucocorticoid hormone therapy is not pathogenetic for AA. The use of glucocorticoid hormones is advisable only for the prevention of early (anaphylaxis, fever, chills, rashes) and late complications (serum sickness) of ATG** therapy within 14–21 days from the start of the ATG** course.

• In patients with AA, when indications are present, it is recommended to administer replacement transfusion therapy with blood components [12,16,35].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: treatment with horse ATG** and #CsA** is impossible without modern replacement transfusion therapy with blood components. First of all, this concerns the use of red blood cell suspension and platelet concentrate, the application algorithm of which is determined by the severity of the disease course, i.e., the severity of anemic and hemorrhagic syndromes and the stage of therapy.

To relieve anemic syndrome in AA, a red blood cell suspension cleared of leukocytes and platelets should be used, taking into account the donor and recipient erythrocyte phenotype. For a single transfusion of donor platelets, the following calculation is used: 1 dose of platelet concentrate (0.5–0.7×10¹¹ platelets) per 10 kg of the patient's body weight, obtained from a single donor. Transfusions are performed before each administration of ATG**, and subsequently depending on the blood platelet count and the presence and severity of hemorrhagic syndrome. 

3.4 Supportive care for patients with AA.

• In all patients under 18 years of age with AA during treatment, upon the development of anemic syndrome, bleeding, or high risk of hemorrhagic syndrome, it is recommended to provide blood product transfusion support (see Appendix A3.3) [106-108].

GCR CLOE 5Grade of Recommendation C (Level of Evidence – 5)

Comments: Transfusion support is carried out in accordance with Order of the Ministry of Health of the Russian Federation No. 1170n dated October 28, 2020, "On Approval of the Rules for Providing Medical Care to the Population in the Field of Transfusiology", registered with the Ministry of Justice of the Russian Federation on November 27, 2020, No. 61123, which regulates the procedure for transfusion support of patients in need of such medical care. As an additional guide, methodological guidelines and textbooks describing the algorithm for selecting blood components, dose calculation, and indications for blood transfusion can be used [111].

• In all patients under 18 years of age with AA during intensive therapy, it is recommended to place an implantable infusion/injection port/catheter*** [112-114].

Level CLevel 5Level of recommendation evidence C (reliability level of evidence – 5).

Comments: the presence of a central venous catheter providing the ability to monitor central venous pressure, frequent blood sampling, and high fluid administration rates is absolutely necessary at the initial stages of therapy and in high-risk patients receiving intensive treatment, including HSCT.

Comments: modification of antimicrobial therapy is performed based on clinical symptoms, instrumental, and microbiological test results. If fever persists and patients are clinically stable, it is acceptable to await a clinical response to initial antibacterial therapy within 48–72 hours, as fever is not the sole indicator of the severity of the infectious process. The prescription of carbapenems as first-line systemic antibacterial drugs for febrile neutropenia is justified in patients with severe infections, involvement of the abdominal or perirectal regions, and the development of sepsis.

Comment Due to the fact that high-risk patients often receive prophylaxis with drugs active against molds, it is necessary to take into account the drug type, dose, route of administration, and, if possible, serum concentration. If the patient received the oral form of these drugs and the blood concentration was therapeutic, they should be discontinued and empirical therapy with other systemic antifungal drugs (caspofungin**, micafungin**, anidulafungin, or Amphotericin B [liposomal]) should be prescribed. In cases where the concentration was subtherapeutic, the oral drug forms should be discontinued, and intravenous voriconazole** should be prescribed to achieve an effective concentration. If persistent fever continues despite achieving a therapeutic blood concentration of voriconazole**, one should switch to the empirical antifungal therapy strategy. Empirical therapy should be continued until the patient recovers from agranulocytosis [148-151]. Amphotericin B** deoxycholate is not used in empirical therapy in children.

• In patients with AA, the pre-emptive antifungal therapy strategy is recommended in clinics with the capability to immediately perform a full range of diagnostic procedures (chest CT, bronchoscopy followed by BAL fluid examination, and biopsy if necessary) and is performed in all AA patients with febrile neutropenia and examination findings indicating invasive mycosis [123,147-149].

LOE 5Grade of Recommendation C (Level of Evidence – 5). 

• In all patients with AA who develop candidemia/invasive candidiasis, it is recommended to prescribe systemic antifungal therapy [137,138,147-150]:

LOE 5Grade of Recommendation C (Level of Evidence – 5).

Comments on antifungal therapy

- Caspofungin** at a dose of 70 mg/m² – day 1 (maximum 70 mg/day), then 50 mg/m²/day (maximum 50 mg/day) in children over three months of age

- Micafungin** 100 mg once daily intravenously or 2–4 mg/kg/day (for infants in the first month of life, the dose may be increased up to 10 mg/kg/day)

- Anidulafungin at a dose of 200 mg on the first day, followed by 100 mg once daily or 3 mg/kg on the first day, then 1.5 mg/kg/day for children older than one month (drug of choice for patients with hepatic impairment)

- Amphotericin B [liposomal] 3 mg/kg/day (for children older than 1 month)

- Voriconazole** (for children under 12 years and weighing less than 40 kg, loading dose of 18 mg/kg/day in 2 divided doses on the first day, then 16 mg/kg/day in 2 divided doses; for those older than 12 years, 12 mg/kg/day in 2 divided doses on the first day, then 8 mg/kg/day in 2 divided doses. Voriconazole** therapy must always be initiated with the intravenous formulation);

- Amphotericin B [lipid complex] 5 mg/kg/day;

• In all patients with AA and neutropenia and/or hemodynamic instability, Fluconazole** is not recommended for prescription in [148] .

LOE 5Grade of Recommendation C (Level of Evidence – 5). 

• Upon the development of invasive aspergillosis, immediate initiation of intravenous antifungal therapy is recommended for all patients with AA [50–56].

Level of recommendation 5 (Level of evidence – C).

• For children with AA older than two years, voriconazole** is recommended as a first-line drug (therapy is always initiated with intravenous formulations, followed by a switch to the oral route once the process stabilizes) [148, 149, 153].

LOE 4 Level of recommendation C (Level of evidence – 4).

• All patients with AA are recommended to undergo therapeutic drug monitoring of serum voriconazole** concentration and maintain it within the range of 1.0–4.0 mcg/ml [195, 196];

LOE 5 Level of recommendation C (Level of evidence – 5).

• #Isavuconazole at a loading dose of 10 mg/kg 3 times/day for two days (maximum 200 mg), followed by 10 mg/kg/day intravenously or orally for children aged 1 year and older; for children from 6 months to 1 year, the single dose is 6 mg/kg. Monitoring of serum drug concentration is not required. Due to the lack of approved pediatric indications, it is a second-line therapy drug [197, 198];

  • Amphotericin B [liposomal] at a dose of 3–5 mg/kg/day (for children older than 1 month);

  • Amphotericin B [lipid complex] 5 mg/kg/day;

  • Combination therapy: voriconazole**/amphotericin B** + a drug from the other systemic antifungals group (echinocandins: caspofungin**, micafungin**, anidulafungin)

  • Therapy is prescribed for a prolonged period (until the resolution of risk factors) – at least 12 weeks. If invasive aspergillosis develops during prior antifungal prophylaxis with triazole derivatives (voriconazole**, posaconazole**), it is necessary to switch the drug class to Amphotericin B [liposomal] / Amphotericin B [lipid complex]. When fungal involvement of the central nervous system, kidneys, or bones develops, voriconazole** and #isavuconazole are the drugs with the best bioavailability. In the event of renal impairment, administration of the intravenous form of voriconazole** is contraindicated. Upon recurrence of risk factors and a previously confirmed invasive mycosis, secondary prophylaxis must be performed. Voriconazole** must be used. [151-154]

Comments:

• Amphotericin B [liposomal] at a dose of 5–10 mg/kg/day (maximum doses are recommended in case of cerebral involvement; for children older than 1 month);

• Amphotericin B [lipid complex] at a dose of 5–7.5 mg/kg/day;

• #Isavuconazole at a loading dose of 10 mg/kg q8h for two days (maximum 200 mg), followed by 10 mg/kg/day (maximum 200 mg). Therapeutic drug monitoring of serum concentrations is not required. Due to the lack of approved pediatric indications, it is a second-line therapy option [197,198];

• Combination therapy with systemic antifungal drugs may be considered as salvage therapy in extremely aggressive cases of the disease or as second-line therapy: amphotericin B [liposomal] or [lipid complex]** + posaconazole** (in children over 13 years of age) or #isavuconazole and/or caspofungin** (in children over three months of age) [202].

• Surgical intervention, multidisciplinary patient management approach.

• Posaconazole** 15–20 mg/kg/day (up to 800 mg/kg/day in 4 divided doses) in children over 13 years of age. Due to extremely variable pharmacokinetics, therapeutic drug monitoring of serum concentrations is required. The drug is prescribed after the patient stabilizes, including for maintenance treatment and secondary prophylaxis.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

• Patients with AA are recommended medical rehabilitation for complications of the underlying disease and the treatment received [48,49].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: the scope, duration, and nature of rehabilitation measures depend on the patient's age at the time of initial diagnosis and the timing of rehabilitation, the scope of treatment received (immunosuppressive therapy, allo-HSCT, haplo-HSCT, etc.), and comorbid conditions. Rehabilitation of children with aplastic anemia should begin on the first day of the child's illness and continue after treatment completion at all stages of follow-up.

The tasks of the inpatient stage include comprehensive medical rehabilitation aimed at treating and preventing developing motor deficits in patients, habilitation and socialization: individual physical therapy sessions, hydrokinesiotherapy, kinesiotaping, and psychological rehabilitation. Social work and habilitation are carried out as part of rehabilitation, allowing the patient and their legal representatives to become familiar with changing social environment conditions and adapt the patient to environmental conditions, physical and psycho-emotional stress after completing intensive inpatient treatment.

The program involves educators (preschool and school education), social workers, medical psychologists, and physicians of various specialties (medical rehabilitation specialists, neurologists, ophthalmologists, etc.).

Rehabilitation is carried out in accordance with the existing Procedure in 3 stages. The frequency of rehabilitation measures at the 2nd and 3rd stages of rehabilitation depends on the patient's status.

Dispensaergic follow-up (clinical observation) of patients is carried out in accordance with Order of the Russian Ministry of Health No. 629n dated June 10, 2021.

5. Prevention and Dispensary Observation

There are currently no methods for preventing AA.

• All patients with AA are recommended to undergo dispensary follow-up by a hematologist for early diagnosis of relapses, clonal complications, and timely initiation of their treatment [8,9,12].

GDR CLE 5Grade of recommendation C (level of evidence – 5)

Comments: the frequency of monitoring patients with AA after treatment completion is not regulated. During the first year after treatment completion, the patient must be monitored by a hematologist at least once every 3 months. Thereafter, the frequency of monitoring is determined by the hematologist individually, depending on the general condition and well-being of the patient, complications of the therapy received, and the achieved response to therapy, but should not be less than once a year. During follow-up, in addition to physical examination, medical history taking, and symptom assessment, a complete blood count with differential leukocyte count must be performed. Other diagnostic methods may be used at the hematologist's discretion if indicated. To assess clonal evolution to PNH, MDS/AML, the size of the PNH clone in the blood is monitored, and bone marrow aspiration analysis using morphological and cytogenetic examinations is performed once a year.

It should be noted that the patient may have atypical disease manifestations, as well as a combination of a specific disease with other pathologies, which may dictate changes by the attending physician in the algorithm for choosing the optimal diagnostic and treatment tactics.

6. Organization of Medical Care

Indications for planned hospitalization:

1) Administration of a course of ATG** therapy;

2) Bone marrow transplantation;

3) Prophylactic transfusions of donor blood components;

4) Patient evaluation, including biopsies and invasive procedures, when it cannot be performed on an outpatient basis;

5) Elective surgery.

Indications for emergency hospitalization:

1) Development of infectious and/or hemorrhagic complications;

2) Severe thrombocytopenia and/or anemia requiring emergency replacement transfusions.

Indications for patient discharge from the hospital:

1) Following a course of ATG** or bone marrow transplantation – outpatient or short-term inpatient monitoring;

2) Complete management of hemorrhagic and/or infectious complications;

3) Completed set of diagnostic and therapeutic measures.

7. Additional Information

Absent

Criteria for Evaluating the Quality of Medical Care

References

Appendix A1. Working Group Composition

1. Maschan A.A., MD, Professor, Corresponding Member of the Russian Academy of Sciences, Director of the Institute of Hematology, Immunology and Cell Technologies at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health, Russian Society of Pediatric Oncology and Hematology.

2. Novichkova G.A., MD, Professor, Scientific Director of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health, Russian Society of Pediatric Oncology and Hematology

3. Kulagin A.D., MD, Professor, Director of the RM Gorbacheva Research Institute of Pediatric Hematology, Transplantology and Immunology at the First Saint Petersburg State Pavlov Medical University of the Russian Ministry of Health, National Hematological Society.

4. Mikhailova E.A., MD, Professor, Leading Researcher at the Department of Chemotherapy of Hemoblastoses and Hematopoietic Depressions at the National Medical Research Center for Hematology of the Russian Ministry of Health, National Hematological Society.

5. Goronkova O.V., Senior Researcher at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health, Russian Society of Pediatric Oncology and Hematology.

6. Maschan M.A., MD, Deputy General Director for Science at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health, Professor at the Department of Hematology, Oncology and Radiation Therapy at the Pirogov Russian National Research Medical University, Russian Society of Pediatric Oncology and Hematology.

7. Balashov D.N., MD, Head of the Hematopoietic Stem Cell Transplantation Department No. 2 at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health.

8. Bronin G.O., PhD, Head of the Bone Marrow and Hematopoietic Stem Cell Transplantation Department at the Morozov Children's City Clinical Hospital of the Moscow Healthcare Department.

9. Vakhonina L.V., pediatric oncologist, Sverdlovsk Regional Children's Clinical Hospital.

10. Dinikina Yu.V., PhD, Head of the Department of Chemotherapy for Oncohematological Diseases and Bone Marrow Transplantation for Children at the Almazov National Medical Research Centre of the Russian Ministry of Health

11. Zubarovskaya L.S., MD, Professor, Deputy Director for Transplantation, Head of the Department of Pediatric Oncology, Hematology and Transplantology at the RM Gorbacheva Research Institute of Pediatric Hematology, Transplantology and Immunology at the First Saint Petersburg State Pavlov Medical University of the Russian Ministry of Health.

12. Kirgizov K.I., PhD, Deputy Director for Research, Head of the Department of Pediatric Bone Marrow and Hematopoietic Stem Cell Transplantation at the L.A. Durnov Research Institute of Pediatric Oncology and Hematology at the N.N. Blokhin National Medical Research Center of Oncology of the Russian Ministry of Health, Executive Director of the Russian Society of Pediatric Oncology and Hematology.

13. Paina O.V., PhD, Head of the Pediatric Bone Marrow Transplantation Department No. 1 at the RM Gorbacheva Research Institute of Pediatric Hematology, Transplantology and Immunology at the First Saint Petersburg State Pavlov Medical University of the Russian Ministry of Health.

14. Skorobogatova E.V., MD, Head of the Bone Marrow Transplantation Department at the Russian Children's Clinical Hospital, a separate structural unit of the Pirogov Russian National Research Medical University of the Russian Ministry of Health

15. Fechina L.G., PhD, Deputy Chief Medical Officer for Oncology and Hematology at the Sverdlovsk Regional Children's Clinical Hospital, Chief freelance specialist pediatric oncologist of the Sverdlovsk Region.

16. Shelikhova L.N., PhD, Head of the Hematopoietic Stem Cell Transplantation Department No. 1 at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health.

17. Rumyantsev A.G., MD, Academician of the Russian Academy of Sciences, Scientific Director of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Professor at the Department of Hematology, Oncology and Radiation Therapy of the Pediatric Faculty at the Pirogov Russian National Research Medical University of the Russian Ministry of Health.

18. Trakhtman P.E., MD, Head of the Department of Transfusiology, Harvesting and Processing of Hematopoietic Stem Cells at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health

19. Solopova G.G., PhD in Medicine, Deputy Chief Physician for Infection Control, Head of the Infection Control Department, National Medical Research Center of Pediatric Hematology, Oncology and Immunology named after Dmitry Rogachev of the Russian Ministry of Health

Conflict of interest: none declared

Appendix A2. Methodology for Developing Clinical Guidelines

Target audience for these clinical guidelines:

1. Pediatric oncologists-hematologists

2. Hematologists

3. Pediatricians

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 acquired aplastic anemia aged 0 to 18 years.

1. General provisions

Hematopoietic stem cell transplantation (HSCT) is a medical technology used to treat 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 chemotherapy and radiation therapy; 2) replacement of the bone marrow hematopoietic function with donor hematopoietic stem cells; and 3) immunological (alloimmune) effect of donor immunocompetent cells on the recipient's residual lymphohematopoiesis.

Allogeneic HSCT is the standard first-line therapy for high-risk acquired aplastic anemia (AAA), as well as for relapsed and refractory forms of AAA.

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 in the early post-engraftment phase (days 30–100)

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

3. Selection of donor and HSC source [50]

To ensure timely donor selection, all patients with AAA, upon establishing indications for HSCT, undergo tissue (HLA) typing of the patient and potential related donors. The strategy for family member typing is determined by the clinic's policy regarding the use of HSC donors.

A donor for a patient with AAA can be:

a. Fully matched related donor

b. Related partially compatible (haploidentical) donor

c. Unrelated compatible donor or unrelated partially compatible donor with an acceptable degree of mismatch

d. Unrelated umbilical 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)

4. Patient evaluation prior to HSCT [51]

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 the evaluation of the patient prior to HSCT:

• 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), determination of antibodies to erythrocyte antigens in blood serum, determination of antibodies to blood group antigens, determination 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

• A26.07.004 Microbiological (culture) examination of oral mucosa discharge for non-spore-forming anaerobic microorganisms;

• A26.01.010 Microbiological (culture) examination of skin scraping for fungi (yeast, molds, dermatophytes).

• 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)

• A26.09.011 Microbiological (culture) examination of lavage fluid for aerobic and facultatively anaerobic microorganisms.

• A26.10.004 Microbiological (culture) examination of biopsy specimen for mycelial fungi.A26.09.030 Microbiological (culture) examination of bronchoalveolar lavage fluid for fungi (yeast and mycelial).

  A26.02.001 Microbiological (culture) examination of wound discharge for aerobic and facultatively anaerobic microorganisms.

  A26.19.001 Microbiological (culture) examination of feces/rectal swab for dysentery agent (Shigella spp.);

  A26.19.003 Microbiological (culture) examination of feces/rectal swab for microorganisms of the genus Salmonella (Salmonella spp.);

  A26.19.004 Microbiological (culture) examination of feces/rectal swab for Yersinia (Yersinia spp.);

  A26.19.004.001 Microbiological (culture) examination of feces/rectal swab for the causative agent of yersiniosis (Yersinia enterocolitica);

  A26.19.007 Microbiological (culture) examination of feces/rectal swab for the causative agent of Clostridioides difficile infection (Clostridium difficile);

• A26.19.078 Microbiological (culture) examination of feces/rectal swab for diarrheagenic Escherichia (EHEC, EPEC, ETEC, EAgEC, EIEC). Examination of calcium level in urine

• Urine phosphorus assay

• A09.28.067 Examination of chloride level in urine.

• 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 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)

• 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

• Otorhinolaryngologist

• Neurologist

• Dentist

• Medical psychologist

• Cardiologist

• Pulmonologist

• Endocrinologist

• Obstetrician-gynecologist

5. Donor evaluation prior to HSCT [52]

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 the evaluation of the donor prior to HSCT:

• 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: determination of hepatitis B virus surface antigen (HBsAg) in blood, determination of antibodies to hepatitis B core antigen (HBcAg) in blood, molecular biological testing of blood for antibodies to hepatitis C virus in blood, molecular biological testing of blood for antibodies to Treponema pallidum in blood, determination of immunoglobulin M and G (IgM, IgG) antibodies to human immunodeficiency virus HIV-1 in blood, determination of immunoglobulin M and G (IgM, 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 the evaluation of the donor prior to HSCT:

• Targeted chest X-ray (2 projections)

• 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, 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)

6. Graft procurement [53]

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

Donor BM procurement [54]

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 devices required for donor BM procurement:

• Gelatin** – 4% solution

• Rocuronium bromide**

• 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

Donor HSPC procurement [55]

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 devices required for donor HSPC procurement:

• 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

• Sodium heparin**

• Packed red blood cells

• Blood transfusion sets

• Discofix (three-way stopcock)

• Empty sterile blood component bag

Cord blood procurement [56]

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 [57]

Graft processing is a set of technical manipulations aimed at modifying the qualitative and quantitative parameters of the HSC graft in order to ensure optimal functional characteristics of the graft 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 [58]:

• 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 thereof) are necessary when the procedures of procurement, processing, and administration of the graft are separated in time and space. Transportation of the HSC graft can be carried out in a native state and after cryopreservation. Native graft transportation is carried out at a temperature range of +3 to +8^oC while maintaining the cold chain. The maximum transportation and storage period (including processing) without cryopreservation is 72 hours from the moment of HSC graft procurement. Cryopreserved graft transportation is carried out at a temperature range of -150 to -196^oC while maintaining the cold chain.

7. Conditioning regimen [59]

The conditioning regimen is a program of immunosuppressive and chemotherapy +/- radiation therapy, the purpose of which is the eradication of the recipient's hematopoietic and immune system and the creation, thereby, of the conditions necessary for the engraftment and functioning of donor hematopoiesis and the formation of the donor immune system.

The conditioning regimen includes chemotherapeutic drugs with pronounced immunosuppressive and/or myelosuppressive effects. The set of drugs, doses, and sequence of administration of chemotherapeutic agents within the conditioning regimen may vary. The choice of the conditioning regimen is determined by the HSCT technological platform implemented in the transplant center and the baseline characteristics of the donor-recipient pair.

Medicines used in the conditioning regimen in AAA (total course dose) [59, 169]

• #Treosulfan, 30-42 g/m², intravenous [60]

• #Busulfan**, 8 mg/kg, oral form [61,155,156]

• #Fludarabine**, 100-180 mg/m², intravenous [62,63]

• Thiotepa, 5-10 mg/kg, intravenous [157]

• #Melphalan** 140 mg/m², intravenous [157]

• #Cyclophosphamide**, 40-200 mg/kg, intravenous [63]

• ATG: Equine anti-thymocyte globulin at a dose of 20 mg/kg/day for 5 days, course dose 100 mg/kg, intravenous [157]

  Anti-thymocyte globulin** (Rabbit) 5 mg/kg, intravenous [157]

• Total body irradiation 2-6 Gy [159]

8. Introduction to GVHD [64]

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) Classical aGVHD: a clinical syndrome developing within up to 100 days after HSCT or donor lymphocyte infusion (DLI). The underlying mechanism is acute inflammatory tissue damage and organ dysfunction. Classical target organs: skin, gastrointestinal tract (GIT), liver. Skin involvement manifests as maculopapular rash, epidermolysis. GIT involvement manifests as diarrhea, nausea, vomiting, hemocolitis, ileus. Liver involvement manifests as 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.

9. GVHD prophylaxis

The goal of GVHD prophylaxis is to prevent the development of GVHD overall and, in particular, 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 the section Graft processing) and on pharmacological intervention in the recipient. The list of drugs used in GVHD prophylaxis is presented below [65]. Drugs are grouped into GVHD prophylaxis regimens; the choice of regimen is determined by the GVHD risk in the donor-recipient pair and the HSCT technological platform implemented at the transplant center.

#Ciclosporin**, 3 mg/kg/day, intravenously [160-164]

#Methotrexate**, 10-15 mg/m²/day, intravenously [160-164]

• #Tacrolimus**, 0.02-0.03 mg/kg/day, orally or intravenously [160,163,164,165]

• #Mycophenolate mofetil**, 30 mg/kg/day, orally [163,164,166-168]

• #Cyclophosphamide**, 50 mg/kg/day, intravenously [67,163,164,166,169,170]

• Antithymocyte globulin**, rabbit 1.5-2.5 mg/kg/day, intravenously, total dose 2.5-10 mg/kg [68,163,164,169,171]

• #Bortezomib**, 1.3 mg/m²/day, intravenously [69,163,164,172]

• #Abatacept**, 10 mg/kg (not exceeding 800 mg), intravenously, [70,157,163,164,173]

• #Tocilizumab**, 4-8 mg/kg, intravenously once a week [71,163,164,174]

• #Sirolimus 4-12 mg/day, orally [72,163,164,175,176]

• #Everolimus** 1.5 mg/m² twice daily, orally [73,163,164]

• #Rituximab**, 375 mg/m²/week, intravenously [74,163,164,174,177]

10. Graft engraftment (myeloreconstitution) [4]

The first stage in evaluating HSCT efficacy is achieving graft engraftment. Graft engraftment criteria include: achieving a leukocyte concentration of more than 1 thousand/µL, neutrophil count of more than 0.5 thousand/µL, and platelet count above 20 thousand/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, when using certain transplantation technologies (e.g., post-transplantation cyclophosphamide or double-unit umbilical cord blood transplantation), engraftment may occur at a later date. Opportunistic viral infections may also delay graft engraftment.

11. Diagnosis and therapy of acute GVHD [75]

The diagnosis of aGVHD is based on the observation and recording of characteristic clinical manifestations and symptoms in the recipient after HSC graft infusion. To verify the clinical diagnosis of aGVHD, a biopsy of the involved organ and histological examination are required. The severity of aGVHD is assessed in accordance with the international grading system given in Tables 1 and 2 [75].

Upon establishing the clinical diagnosis of aGVHD, therapy is initiated, the composition of which is determined by the severity of aGVHD. The severity of aGVHD and the response to first-line therapy determine the prognosis of aGVHD progression and survival. If there is no 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 therapy of GVHD [75]:

• Complete blood count (CBC) with differential

• General clinical biochemical blood 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, 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 blood

• Qualitative PCR detection of Cytomegalovirus DNA in peripheral blood

• Qualitative PCR detection of human herpesvirus type 6 (HHV-6) DNA in peripheral 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), determination of antibodies to erythrocyte antigens in blood serum, determination of antibodies to blood group antigens, determination 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: detection of Hepatitis B virus surface antigen (HBsAg) in blood, detection of antibodies to Hepatitis B virus core antigen in blood, detection of antibodies to Hepatitis C virus in blood, detection of antibodies to Treponema pallidum in blood, 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

• A26.07.004 Microbiological (culture) examination of oral mucosa discharge for non-spore-forming anaerobic microorganisms;

• A26.01.010 Microbiological (culture) examination of skin scraping for fungi (yeast, molds, dermatophytes).

• 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)

• A26.09.011 Microbiological (culture) examination of lavage fluid for aerobic and facultatively anaerobic microorganisms.

• A26.10.004 Microbiological (culture) examination of biopsy specimen for mold fungi A26.09.030 Microbiological (culture) examination of bronchoalveolar lavage fluid for fungi (yeast and mold). A26.02.001 Microbiological (culture) examination of wound discharge for aerobic and facultatively anaerobic microorganisms.

• A26.19.001 Microbiological (culture) examination of feces/rectal swab for dysentery agent (Shigella spp.);

• Urine calcium assay

• Urine phosphorus assay

• A09.28.067 Examination of chloride level in urine.

• 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 therapy of GVHD [75]:

• 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 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 therapy of GVHD (daily dose) [75–77,87,164]:

• #Methylprednisolone**, 2 mg/kg/day, intravenously [178]

• Antithymocyte globulin** rabbit-rabbit 1.5-2.5 mg/kg/day intravenously, total dose 2.5-10 mg/kg [68]

• Ciclosporin**, 1-3 mg/kg/day, orally

• #Methotrexate**, 5-15 mg/m²/day, intravenously [78]

• #Tacrolimus**, 0.03 mg/kg/day, orally [66]

• #Mycophenolate mofetil**, 190-1600 mg/m²/day, orally [79,178]

• #Alemtuzumab**, 0.3-2 mg/kg intravenously (total dose administered over 2-6 days) [80,178]

• Ibrutinib**, 140-420 mg/day, orally [178]

• #Ruxolitinib**, 2.5-10 mg/m² twice daily, orally [81,178]

• #Etanercept**, 0.4 mg/kg, subcutaneously twice a week [82,115,178]

• #Bortezomib**, 1.3 mg/m²/day, intravenously [69,178]

• #Abatacept**, 10 mg/kg (not exceeding 800 mg), intravenously every 2 weeks for the first 3 doses, then every 4 weeks [70,115,178]

• #Tocilizumab**, 4-8 mg/kg, intravenously once a week [71,115,178]

• #Sirolimus 2-4 mg/day, orally [72,115,178]

• #Everolimus** 1.5 mg/m² twice daily, orally [73,178]

• #Infliximab**, 10 mg/kg, intravenously once a week [83,178]

• #Rituximab**, 375 mg/m²/week, intravenously [74,178]

• Topical glucocorticoids (ATC code D07A "Corticosteroids")

• Topical calcineurin inhibitors (from the group "Other dermatological preparations", ATC code D11AH) – #tacrolimus, #pimecrolimus** in topical form [84,85]

• Mesenchymal stromal cells

• #Imatinib**, 65-260 mg/m²/day, orally [86]

12. Diagnosis and therapy of chronic GVHD [87]

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 severity of organ function impairment (see Table 4) [89].

• 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. –

* ALP may be elevated in growing children without reflecting hepatic dysfunction

Abbreviations: KI – Karnofsky Index, LI – Lansky Index, BSA – body surface area, DA – daily activities, 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.

13. Prevention and Management of Infectious Complications [90]

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 [91]

The main risk factors for bacterial infections in HSCT recipients in the early period include granulocytopenia, use of central venous catheters, mucositis, dermatitis, preexisting colonization with multidrug-resistant pathogens, and refractory underlying disease. Early bacterial infections are caused by Gram-positive cocci and Gram-negative bacilli. In the late period, the main risk factors are humoral immune deficiency and hyposplenism, predisposing to infections with encapsulated microorganisms (Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis). The main types of bacterial infections after HSCT are pneumonia, sinusitis, enterocolitis, 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 [92]

The main risk factors for viral infections in HSCT recipients are lymphopenia, cellular immunodeficiency, ongoing GVHD, systemic corticosteroid therapy, lymphodepleting monoclonal antibody therapy, use of mismatched and partially compatible donors, presence of persistent viral infection in the patient and/or donor prior to HSCT, ex vivo T-cell depletion, and refractory underlying disease. The major 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 generalized 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 [93,94]

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.

14. Prevention of Specific (Organ-Specific) Complications

a. Hemorrhagic Cystitis [95]

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.

b. Hepatic Veno-Occlusive Disease (Sinusoidal Obstruction Syndrome) [96,97]

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 for 3 days or >2 mg/dL over 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.

c. Mucositis [98]

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.

d. Posterior Reversible Encephalopathy Syndrome (PRES) [99]

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.

e. Respiratory Distress Syndrome [100]

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.

f. Thrombotic Microangiopathy [101]

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 damage 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 others), fungal infections, and GVHD. Therapy for TA-TMA includes dose reduction or discontinuation of calcineurin inhibitors, syndromic supportive care, and targeted pharmacotherapy (see below).

15. Nutritional Support [102]

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.

16. Transfusion Therapy [103]

During HSCT, the recipient's hematopoiesis is replaced by donor hematopoiesis. This process involves transient hematopoietic aplasia, lasting from several days to several weeks. Hematopoietic aplasia, by definition, develops in 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 the prevention of transfusion-associated graft-versus-host disease (TA-GVHD) is gamma irradiation of blood components at a dose of 25 Gy [104].

Appendix B. Physician Action Algorithms

Appendix C. Patient Information

Aplastic anemia (AA) is a condition in which the patient's bone marrow stops producing sufficient quantities of all major blood cell types – red blood cells, white blood cells, and platelets. Hence the name of the disease: it combines anemia (insufficient red blood cell count, low hemoglobin level) and hematopoietic aplasia (suppression of all blood cell production).

Aplastic anemia is a rare disease, with an incidence of about 2–6 cases per million population per year. AA can occur in both children and adults; it is believed that peak incidence occurs in young adulthood (15–30 years) and then in the elderly (over 60 years). Men and women are affected with equal frequency.

In the vast majority of cases, the cause of the disease is unknown – this is referred to as idiopathic AA. However, sometimes the disease is associated with certain congenital disorders or external factors: the use of certain medications (quinine, chloramphenicol, etc.), work with toxic substances (herbicides, insecticides, certain solvents including benzene), radiation, or prior infections (hepatitis viruses, Epstein-Barr virus, HIV; possibly also cytomegalovirus).

It is believed that idiopathic AA typically has an autoimmune origin, meaning it occurs when the body's immune system for some reason begins to attack its own bone marrow cells.

Manifestations of aplastic anemia are associated with pancytopenia – a deficiency of all types of blood cells.

• Erythrocyte deficiency – paleness, weakness, shortness of breath, palpitations, dizziness, headaches.

• Platelet deficiency – bleeding, including nasal and gingival bleeding; appearance of bruising and petechiae (small subcutaneous hemorrhages).

• Leukocyte deficiency – low resistance to infections. Infectious diseases that are poorly responsive to therapy or frequently recurring may occur.

Aplastic anemia varies in severity: mild, moderate, severe, and very severe forms of AA are distinguished.

AA can be suspected if a child presents with the listed complaints and symptoms, and confirmed based on complete blood count results, which show a sharp decrease in erythrocyte, leukocyte, and platelet counts. Other clinical and laboratory studies are also performed, but the definitive diagnosis is established only by analyzing a bone marrow sample obtained via aspiration and trephine biopsy. Bone marrow examination is necessary to rule out other possible causes of blood cell deficiency, such as leukemia, myelodysplastic syndromes, myelofibrosis, etc.

In aplastic anemia, bone marrow examination indicates aplasia or hypoplasia of hematopoiesis – meaning the bone marrow practically does not produce blood cells or produces them in significantly fewer numbers than required.

During diagnostic evaluation, it is also essential to reliably distinguish AA from congenital Fanconi anemia, as the treatment for these diseases (including bone marrow transplantation protocols) differs markedly. Special tests (such as the DEB test) may be used to confirm or exclude Fanconi anemia.

Treatment of severe and very severe AA must be initiated immediately after diagnosis, as the condition of severe deficiency of all blood cells is life-threatening.

If the development of AA is caused by a specific external factor (drugs, radiation), this factor must be eliminated first. However, as already mentioned, an external triggering factor for the disease can be identified in only a very small number of cases.

Since AA is presumed to be autoimmune in nature, immunosuppressive therapy is widely used for its treatment – that is, therapy aimed at suppressing the immune system, which «attacks» bone marrow cells. Typically, such therapy includes antithymocyte globulin** (ATG**) combined with #ciclosporin**; systemic corticosteroids may be used to prevent side effects of ATG**. Response to this therapy usually develops slowly: in successful cases, the bone marrow gradually recovers a few weeks or months after drug administration and begins producing healthy cells.

Hospitalization is generally required to administer a course of immunosuppressive therapy with ATG**. The main complications during treatment are infections and bleeding. If there is no response to the first course of therapy, a repeated course may be administered. Immunosvuppressive therapy is effective in approximately 70% of cases. Unfortunately, some patients experience relapses of the disease afterward.

Allogeneic bone marrow transplantation, if successful, leads to a complete cure. Transplantation is particularly preferred when the patient is young and has a fully matched related donor (brother or sister). In the absence of a related donor, the use of a matched unrelated donor or a haploidentical related donor (haploidentical transplantation) is acceptable. Previously, it was believed that transplants from unrelated or partially matched donors in AA carried very high risks, but their outcomes have improved significantly now.

During the treatment of AA, intensive supportive care with blood components (transfusions of donor platelets, red blood cells, and sometimes granulocytes in severe infections) is often required. However, if bone marrow transplantation is planned, it must be kept in mind that multiple transfusions prior to transplantation increase the likelihood of graft rejection.

Prevention and treatment of bacterial, viral, and fungal infections, which pose a major danger to AA patients, are very important. Effective combinations of systemic antibacterial and systemic antifungal drugs are used for treatment, sometimes over a long period.

Growth factors – medications that help the bone marrow produce necessary cells – are also used as adjunctive therapy. These include granulocyte colony-stimulating factor, which stimulates leukocyte production, and erythropoietin-group drugs (ATC group B03XA), which stimulate erythrocyte production.

Without treatment, patients with severe forms of aplastic anemia die within a few months. However, with modern adequate treatment, the prognosis is quite good.

As already mentioned, immunosuppressive therapy is effective in many cases. Patients may feel well for a long time after such therapy, but some experience a relapse (return) of the disease. Regular check-ups, primarily blood tests, are necessary for the timely detection of relapse.

Bone marrow transplantation from both related and unrelated donors is associated with certain risks, primarily immune and infectious complications. However, if the transplantation is successful, complete remission and cure are achieved.

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

Appendix G1. Eastern Cooperative Oncology Group (ECOG) Performance Status Scale

Original title: The ECOG Scale of Performance Status

Source: Oken M.M. et al. Toxicity and response criteria of the Eastern Cooperative Oncology Group. Am. J. Clin. Oncol. 1982;5(6):649–65 [105]

Type: rating scale

Purpose: clinical assessment of the patient's general condition

Content and interpretation: