Autoimmune hemolytic anemia: Clinical Guidelines

List of Abbreviations

AIHA – autoimmune hemolytic anemia

ALPS – autoimmune lymphoproliferative syndrome

ALT – alanine aminotransferase

AST – aspartate aminotransferase

APS – antiphospholipid syndrome

IVIG – intravenous immunoglobulin (normal human immunoglobulin**)

HIV – human immunodeficiency virus

GCH – giant cell hepatitis

HUS – hemolytic-uremic syndrome

DIC – disseminated intravascular coagulation

DNA – deoxyribonucleic acid

CID – combined immunodeficiency

CT – computed tomography

MMF – mycophenolate mofetil**

MRI – magnetic resonance imaging

CVID – common variable immunodeficiency

CR — complete response

PCH – paroxysmal cold hemoglobinuria

SLE – systemic lupus erythematosus

ESR – erythrocyte sedimentation rate

FES – Fisher-Evans syndrome

HSCT – hematopoietic stem cell transplantation

TTP — thrombotic thrombocytopenic purpura

US – ultrasound examination

CAD – cold agglutinin disease

CAS – cold agglutinin syndrome

CMV — cytomegalovirus

ALP – alkaline phosphatase

PR — partial response

EBV – Epstein-Barr virus

ANA – antinuclear antibodies

CBC – complete blood count

CD – cluster of differentiation, nomenclature of human leukocyte differentiation antigens

CTLA-4 – cytotoxic T-lymphocyte associated protein 4, encoded by the gene of the same name

IgG, A, M, E – immunoglobulins class G, A, M, E

IPEX – immune dysregulation polyendocrinopathy enteropathy X-linked syndrome

LRBA – lipopolysaccharide-responsive and beige-like anchor protein

MCV – mean corpuscular volume

NGS – next generation sequencing

NIPA – non-immune protein adsorption

PI3KD – gene encoding phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta

Rh – Rhesus factor

STAT1-GOF – gain-of-function mutation in the STAT1 gene (encoding signal transducer and activator of transcription 1)

STAT3-GOF – gain-of-function mutation in the STAT3 gene (encoding signal transducer and activator of transcription 3)

TCR – T-cell receptor

VZV – varicella zoster virus

WAS – gene encoding Wiskott-Aldrich syndrome protein

Terms and Definitions

Autoimmune hemolytic anemia is an acquired form of hemolytic anemia in which autoantibodies bind to erythrocyte membrane antigens, causing their destruction.

Alloantibodies are antibodies directed against erythrocyte antigens, primarily the Rhesus factor, which appear following transfusion of incompatible blood or during pregnancy.

Autoantibodies are antibodies produced by the immune system against a person's own antigens.

Extravascular (intracellular) hemolysis is the destruction of erythrocytes mediated by the mononuclear phagocyte system in the spleen or liver.

Intravascular hemolysis is the destruction of erythrocytes within the general vascular network.

Paroxysmal cold hemoglobinuria (PCH) is an autoimmune hemolytic anemia caused by antibodies that react most avidly with erythrocytes at low temperatures, causing complement activation with predominantly intravascular hemolysis. PCH is characterized by the presence of Donath-Landsteiner antibodies.

“Warm” autoimmune hemolytic anemia is AIHA characterized by the production of IgG autoantibodies (less commonly IgM, IgA) that interact with erythrocyte antigens at 37°C without causing complement activation.

Cold agglutinin disease is an autoimmune hemolytic anemia with a strongly positive direct Coombs test (direct antiglobulin test) for anti-C3d (and negative or weakly positive for anti-IgG) and a cold agglutinin titer of 1:64 or higher, interacting with erythrocytes at 4°C. Patients typically have a clonal B-cell lymphoproliferative disorder detectable in the blood or bone marrow, but without clinical or radiological features of malignancy. It is extremely rare in children.

Cold agglutinin syndrome is an autoimmune hemolytic anemia with a strongly positive direct Coombs test (direct antiglobulin test) for anti-C3d (and negative or weakly positive for anti-IgG) and a cold agglutinin titer of 1:64 or higher, interacting with erythrocytes at 4°C. CAS develops secondarily in the setting of an autoimmune disease, infection, lymphoproliferative disorder, or malignancy.

Cold agglutinins are IgM autoantibodies directed against I/i system erythrocyte antigens.

1. Brief information on the disease or condition

1.1 Definition of the disease or condition

Autoimmune hemolytic anemia (AIHA) is an acquired form of hemolytic anemia in which autoantibodies bind to erythrocyte membrane antigens, causing their destruction (hemolysis). Hemolysis triggers a compensatory production of erythrocytes due to increased erythropoietin levels; however, this response is usually insufficient to restore normal blood hemoglobin, resulting in anemia [1]. AIHA can be idiopathic (primary) or secondary, depending on the presence of an underlying disease or condition predisposing to immune dysregulation. AIHA is also subdivided into "warm" and "cold" forms depending on the optimal temperature of antibody binding [1, 2].

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

From an etiological perspective, AIHA can be divided into idiopathic (37%) and secondary (63%), with the latter being a component or characteristic feature of an underlying condition associated with immune dysregulation. In children, such conditions most commonly include autoimmune syndromes, inborn errors of immunity, and infections [3].

Less frequent etiological factors include malignancies, previous blood component transfusions, organ transplantation (especially when tacrolimus** is used for immunosuppression), and medications [1]. The main etiological factors are presented in Table 1 [4].

Table 1. Most common underlying conditions associated with AIHA in children.

The cause of autoimmune hemolytic anemia (AIHA), like any autoimmune process, is a "breakdown" of immunological tolerance to self-antigens. The mechanism of this process has not yet been fully elucidated. Since AIHA is a rare disease, it is possible that the predisposition to such disorders is partly due to genetic defects [5].

It has been shown that in AIHA, as in many other autoimmune disorders, there is a suppression of the suppressor function of T lymphocytes, which promotes an increase in the population of B lymphocytes that produce antibodies against self-structures [5]. The breakdown of tolerance occurs when it is established in the presence of small amounts of antigen. Tolerance developed after contact with a large amount of antigen practically never disappears. As an example, the absence of autoaggression against ABO system antigens is usually cited. Different forms of AIHA may have different underlying leading causes. There is evidence of the detection of antilymphocyte antibodies and antilymphocytotoxins in various autoimmune diseases. In B-cell tumors, autoantibodies are products of the tumor clone.

Erythrocyte destruction in most cases is associated with the production of IgG class antibodies, less frequently IgA and IgM; complement may also be involved, which determines both the pathophysiology and treatment of AIHA [1].

The properties of autoantibodies determine the features of various forms of AIHA. Incomplete autoagglutinins cause erythrocyte agglutination only in an aqueous-saline medium, whereas the action of complete ones manifests in any medium [6, 7]. Incomplete warm autoantibodies are fixed on erythrocytes and interact through immunoglobulin Fc fragments with macrophage Fc receptors, resulting in the loss of a portion of the erythrocyte membrane, a change in its biophysical state, and, above all, the properties of ion channels. This leads to the formation of microspherocytes and a sharp acceleration of erythrocyte destruction (sequestration) in the spleen and sometimes in the liver [8]. Cold agglutinins cause transient agglutination of erythrocytes in the cold, leading to the fixation and activation of complement on their surface with subsequent membrane damage. Hemolysins activate complement, which damages the membrane within the vascular bed. In this form of AIHA, the infectious agent may share similarities with erythrocyte antigenic structures or modify them slightly at the initial stage, subsequently leading to autosensitization [9].

Drugs can cause AIHA both with and without impaired immune regulation. The number of drugs that can cause drug-induced immune hemolytic anemia has increased over the past few decades (currently around 125) [10]. Three groups of drugs predominate: 42% are systemic anti-infectives, 15% are anti-inflammatory and antirheumatic drugs, and 11% are antineoplastic drugs. Cephalosporins have become the most common group of medications causing AIHA, accounting for 70% of cases [10]. The mechanisms and examples of drug-induced AIHA are described below and in Table 2 [10].

Table 2. Mechanisms and examples of drug-induced AIHA.

¹ – Not registered in the Russian Federation.

There are three general mechanisms explaining the development of drug-induced AIHA:

• Antigen modification. In this case, the drug interacts with a specific target protein on the erythrocyte surface, altering its primary structure. As a result, circulating lymphocytes identify this protein as foreign and produce antibodies against it. These antibodies, in turn, interact with unchanged copies of the protein on the erythrocyte surface, subsequently leading to erythrocyte destruction. It should be noted that the drug in this case is not part of the antigenic epitope and is not required for subsequent antibody production, so discontinuation of the drug does not guarantee the cessation of AIHA in these patients [4].

• Haptenization. A hapten is a small molecule that is not antigenic on its own, but upon attachment to a specific target protein increases the immunogenicity of this protein such that circulating lymphocytes will consider it foreign. The hapten is an important component of the antigenic epitope, unlike the mechanism described above. This mechanism can be divided into two distinct categories:

  - stable (covalent) binding, a classic example of which is benzylpenicillin**, especially when administered intravenously in high doses. Following the administration of benzylpenicillin**, the drug binds tightly to the Rhesus antigen on the erythrocyte surface, leading to the formation of a protein complex. Specific IgG autoantibodies target this haptenized protein complex; in this case, complement is not activated. Given the incorporation of the drug into the epitope, autoantibodies react only with haptenized proteins, so discontinuation of the drug often leads to the cessation of hemolysis [10].

  - with weak drug binding to erythrocyte surface antigens, an epitope is formed that contains both the drug and the erythrocyte protein — in this case, autoantibodies occasionally react with both the haptenized protein and the standalone drug. These reactions are typically mediated by IgM, accompanied by complement activation and intravascular hemolysis [10].

• Nonimmunologic protein adsorption (NIPA): it is hypothesized that under this mechanism, the drug causes morphological changes in the structure of erythrocyte surface proteins in such a way that it nonimmunologically attracts several plasma proteins, including IgG, albumin, C3, fibrinogen, and others. The attachment of polyclonal IgG to the erythrocyte surface shortens their lifespan and can induce opsonization-phagocytosis, leading to extravascular hemolysis. As a rule, complement activation does not occur in this process, since antibodies do not attach immunologically, but rather are adsorbed onto the erythrocyte surface. Classic examples of drugs associated with this mechanism are first-generation cephalosporins and beta-lactamase inhibitors [10].

1.3 Epidemiology of the Disease or Condition

AIHA affects both pediatric and adult populations. In childhood, the disease is rare. Literature data contain information from small series, mostly based on laboratory studies and covering a very small number of centers. The estimated annual incidence based on the French Cohort Study is approximately 0.81 per 100,000 individuals under 18 years of age [11]. The most common form of AIHA in children is associated with "warm" antibodies, diagnosed in more than half of AIHA cases. The exact prevalence of AIHA in childhood is still unknown, but it likely increases with age, as with most autoimmune diseases [1].

1.4 Features of Coding the Disease or Condition

D59.1 Other autoimmune hemolytic anemias

D59.8 Other acquired hemolytic anemias

D59.9 Acquired hemolytic anemia, unspecified

1.5 Classification of the disease or condition

The classification of the disease is based on the thermal properties of the antibodies and immunoglobulin classes. Classically, AIHA is divided into "warm", "cold", and "mixed" types.

"Warm" AIHA is the most common form of immune hemolysis, accounting for 65–70% of cases [3]. It is characterized by the production of high concentrations of IgG class autoantibodies; IgA and monomeric IgM are detected in rare cases. Optimal antibody-antigen interaction occurs at 37°C (body temperature). Autoantibodies are polyclonal and polyspecific, meaning they react with multiple erythrocyte antigens. Hemolysis in this type of AIHA is intracellular, as erythrocytes coated with "warm" antibodies are phagocytosed by splenic macrophages bearing Fcγ receptors. Sometimes splenic macrophages cleave only a portion of the membrane, resulting in the formation of a spherocyte; upon repeated passage through the spleen, the spherocyte is destroyed [12].

Some antibodies have the ability to activate the complement system with the formation of the C3b component; in turn, C3b-opsonized erythrocytes undergo phagocytosis by hepatic macrophages carrying C3 receptors. IgM activate complement very efficiently; IgG1 and IgG3 are also efficient complement activators; IgG2 and IgA are weak complement activators; IgG4 does not activate complement at all. In rare cases, complement activation can lead to the formation of the membrane attack complex (C5b9), resulting in intravascular hemolysis [12].

"Cold" AIHA is caused by the presence of IgM class antibodies. Depending on the etiology, primary cold agglutinin disease (CAD) and secondary cold agglutinin syndrome (CAS) are distinguished [1].

The term CAD is used to designate chronic autoimmune hemolytic anemia caused by IgM class antibodies that bind to erythrocytes at low temperatures. CAD is typically observed in adult patients with a low-grade clonal lymphoproliferative disorder of the bone marrow. The term CAS refers to the secondary form of AIHA, which develops most frequently in children following infections [13].

IgM class autoantibodies bind erythrocyte membrane antigens, typically of the I/i system, at approximately 4°C, although they may retain reactivity up to 30°C, which is defined as a wide thermal amplitude. Thermal amplitude is the highest temperature at which antibodies can bind to erythrocytes and cause their agglutination; the higher this value, the greater the severity of the disease. IgM pentamers fix complement components much more easily than IgG. Activation of the complement cascade occurs when IgM-bound erythrocytes return to the central circulation (reach body temperature), forming C5b9 membrane attack complexes (MAC) that cause intravascular hemolysis. Intracellular hemolysis also occurs with the participation of Kupffer cells in the liver. The pentavalent structure of IgM is capable of binding multiple erythrocytes simultaneously, leading to their agglutination [12, 13].

CAS is primarily associated with infections. Most commonly, this occurs during infection with Mycoplasma pneumoniae and Epstein-Barr virus (EBV), but it can also be associated with other viral infections such as cytomegalovirus (CMV), Varicella, Rubella, parvovirus B19, and hepatitis B. As a result, cold agglutinins are produced and clinically significant hemolysis develops [14, 15]. The initial symptoms usually appear 2 weeks after the onset of the primary infection, and then hemolysis decreases as the infection resolves [1, 4].

Paroxysmal cold hemoglobinuria (PCH) is the most frequent form of AIHA in children. It was originally described by Dressler in 1854. PCH was historically described as a chronic disease in patients suffering from late or congenital syphilis who experienced "paroxysms" of hemoglobinuria after exposure to low temperatures followed by body rewarming. Currently, this disease is extremely rare; more than 50% of described PCH cases are children under 5 years of age with the development of upper respiratory tract infections of various etiologies [16].

The confirmatory test for PCH, which detects the presence of biphasic antibodies, was developed over 100 years ago by Julius Donath and Karl Landsteiner, after whom the pathological antibody is named [17]. Donath-Landsteiner antibodies in PCH are polyclonal IgG class antibodies directed most frequently against the P erythrocyte antigen (83.1%) [16]. They bind to the erythrocyte membrane and fix complement on them at low temperatures in the peripheral parts of the body (extremities), then upon warming (return to the central circulation and reaching body temperature), activation of the full complement cascade occurs, along with intravascular lysis of erythrocytes and the release of free hemoglobin, leading to classic hemoglobinuria.

Clinical and laboratory diagnosis of PCH remains a challenging task due to the rarity of the disease and the complexity of testing methods. The detection of Donath-Landsteiner antibodies requires special blood sample preparation, incubation temperatures, and a long processing time. Recent case reports of PCH noted delayed diagnosis of the disease because it was misdiagnosed as "cold" AIHA [16].

A small number of cases can be classified as "mixed" AIHA; this type reveals serological markers of both "warm" AIHA and CAS [1].

1.6 Clinical presentation of the disease or condition

Clinical manifestations in "warm" AIHA are usually nonspecific and depend on the severity of the anemia. Patients with a subclinical course of hemolysis may be asymptomatic, but moderate-to-severe anemia is most commonly observed in AIHA. About 60% of patients noted clinical manifestations of a nonspecific illness with fever within the month prior to AIHA onset [4].

General symptoms in all types of AIHA:

• Anemic syndrome of varying severity (weakness, decreased exercise tolerance, tachycardia, shortness of breath, dizziness and/or headache);

• Jaundice and/or pallor of the skin and mucous membranes, scleral icterus;

• Dark urine (80%);

• Hepatomegaly (19%), splenomegaly (31%);

In severe anemia, signs of heart failure may be observed (especially with concomitant cardiac pathologies, e.g., heart defects) — rales in the lungs, peripheral edema, jugular venous distention. With the sudden and rapid development of severe anemia, coma and acute renal failure may occur [1, 4].

In addition, patients with secondary AIHA exhibit symptoms associated with the underlying disease; therefore, it is critical to identify signs of potential autoimmune pathology, immunodeficiency, malignancies, and/or infection [4].

In CAD/CAS, in addition to general manifestations, symptoms associated with cooling develop (in up to 90% of patients). Acrocyanosis results from erythrocyte agglutination caused by IgM pentamers in the fingertips, nose, and/or ears upon cold exposure. The purple-gray discoloration and associated pain resolve upon rewarming with minimal reactive hyperemia of the skin. Other cutaneous manifestations of erythrocyte agglutination include a pronounced Raynaud phenomenon, livedo reticularis, urticaria, or very early skin necrosis/ulceration. An episodic worsening of disease manifestations upon cold exposure (including drinking cold water), stress, trauma, surgery, and/or fever is characteristic [1, 4, 13].

2. Diagnostics, medical indications and contraindications for diagnostic methods

Criteria for establishing the diagnosis/condition: the diagnosis is established based on the presence of clinical (pallor, jaundice, hepato/splenomegaly, dark urine) and laboratory manifestations of hemolysis (anemia of varying severity, reticulocytosis, elevated total bilirubin due to the indirect fraction, elevated LDH activity, decreased haptoglobin) and a positive direct Coombs test (direct antiglobulin test) [18].

Confirmation of an AIHA diagnosis requires the presence of hemolytic anemia with positive serology, since 0.1% of blood donors and up to 8% of hospitalized patients may have a positive Coombs test (direct antiglobulin test) without clinical or laboratory evidence of hemolysis [19].

2.1 Complaints and Anamnesis

Comments: in the history of the present illness, attention should be paid to the presence of fever, chills, abdominal/back pain and dark urine, recent infections (ARVI), medication intake or vaccination; recent travel (malaria); open wounds/burns (clostridial septicemia); whether blood transfusions were performed (transfusion reaction); whether intense physical exertion occurred (march hemoglobinuria).

The personal history includes questions about previous episodes of anemia and/or jaundice, episodes of dark urine (especially after taking certain medications, foods, or infections), splenomegaly, cholelithiasis; autoimmune diseases (arthritis, rash, oral ulcers) or immunodeficiency diseases, and infection history (recurrent infections, abscesses, pneumonia, etc.).

It is also necessary to clarify the family history regarding the presence of the above-mentioned manifestations in family members.

2.2 Physical Examination

Comments: assessing the severity of anemic syndrome (tachycardia, tachypnea, tinnitus, gallop rhythm, arterial hypotension, or shock) is crucial for deciding on the urgency of blood transfusion therapy. Examination reveals skin pallor, jaundice (depending on the severity of hyperbilirubinemia), and scleral icterus.

Assessment of lymphoproliferative syndrome via palpation is necessary (possible malignancies, systemic lymphoproliferation), along with checking for acrocyanosis and limb ulcers (symptoms associated with agglutination).

2.3 Laboratory Diagnostics

Comments: the complete blood count (CBC) with differential requires evaluation of the leukocytic and thrombocytic lineages (diagnosis of Evans syndrome). In AIHA, anemia is most commonly macrocytic (increased MCV – mean corpuscular volume). Peripheral blood smear review may reveal microspherocytes (in warm AIHA), polychromasia, and normoblasts (marked normoblastosis can cause pseudoleukocytosis on automated blood analyzers and a left shift in the myeloid series), as well as possible schistocytes (for differential diagnosis with microangiopathies), atypical cells (in leukemias), and erythrocyte agglutination (in CAD/CAS).

In rare cases of warm AIHA, reticulocytopenia may be observed in the early stages of the disease, when antibodies also interact with antigens on the reticulocyte membrane, or in the presence of a concurrent viral infection such as parvovirus B19.

Comments: most patients with hemolysis show elevated bilirubin due to the indirect fraction, increased LDH activity, and decreased serum haptoglobin. Liver function assessment is necessary to identify possible concurrent giant cell hepatitis, especially in infants and young children, or viral liver damage that could have triggered the development of AIHA. Baseline renal evaluation is also required, as the disease itself and its treatment may affect kidney function.

Comments: laboratory diagnostics of AIHA are based on the detection of autoantibodies bound to erythrocytes (direct antiglobulin test / direct Coombs test) and free antibodies in blood serum (indirect antiglobulin test / Coombs test).

The direct antiglobulin test (direct Coombs test) should be performed first using polyspecific sera capable of identifying both IgG and C3 fixed on erythrocytes. If a positive result is obtained, testing with monospecific anti-IgG and anti-C3 sera is required.

Patients with CAD/CAS have a positive result with anti-C3 serum and a negative result with anti-IgG. It is advisable to determine the cold agglutinin titer if the direct antiglobulin test (direct Coombs test) with anti-C3 serum is positive (an antibody titer greater than 1:64 is clinically significant).

In cases of newly diagnosed AIHA in children with intravascular hemolysis, regardless of the direct antiglobulin test (direct Coombs test) results (negative or positive), testing for Donath-Landsteiner autoantibodies must be performed.

In some cases of AIHA, the direct antiglobulin test (direct Coombs test) may be negative (up to 10%). The diagnosis of Coombs-negative AIHA can be established after ruling out all other non-immune-mediated causes of hemolysis (Table 3), and it also requires additional tests, such as using monospecific sera against IgA or IgM.

Table 3. Non-immune hemolytic anemias.

Comments: screening to identify the underlying causes of AIHA is necessary for every child, especially at disease onset at an early age (under 3 years). It should be noted that immunological tests are best performed before starting treatment.

If testing for double-negative T cells (screening for autoimmune lymphoproliferative syndrome, ALPS) is not available, measuring serum vitamin B12 (cyanocobalamin) levels may be considered (significantly elevated in ALPS). Subsequent, more in-depth testing is performed as clinically indicated.

Comments: if CAD is suspected, determination of class M and G antibodies (IgM, IgG) against Mycoplasma pneumoniae in the blood is indicated, which will point to a recent infection.

2.4 Instrumental Diagnostics

2.5 Other Procedures

Comments: in AIHA, the myelogram typically reveals erythroid hyperplasia with no changes in other lineages. Routine bone marrow puncture (obtaining a bone marrow cytological preparation via puncture) is not indicated in children with a typical course of warm AIHA. In adult AIHA patients, bone marrow puncture (obtaining a bone marrow cytological preparation via puncture) is indicated for all patients during initial diagnosis.

When diagnosing CAD, an evaluation to detect clonal lymphoproliferative bone marrow disease is not mandatory to establish the diagnosis. However, if feasible, this evaluation should be performed. Bone marrow examination for the diagnosis of lymphoproliferative disease also includes immunophenotyping of biological material (bone marrow) to identify hemoblastosis markers and pathomorphological examination of bone marrow biopsy (surgical) material (including the use of immunohistochemical methods).

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

Treatment in these clinical guidelines will mainly concern warm AIHA, as the most common form in both adult and pediatric practice. Therapy for cold AIHA is covered within the framework of secondary CAD, since primary CAD does not occur in children. In adult patients, conversely, CAD predominates among cases of cold AIHA.

3.1 Pharmacotherapy

Comments: the total duration of therapy with prednisolone** at a dose of 1-2 mg/kg/day is 3 weeks; thereafter, the dose must be tapered gradually to avoid relapses. Tapering of prednisolone** must always be slow (gradual dose reduction correlates with a lower relapse rate). The side effects of long-term GC use should be taken into account and monitored.

If oral administration of prednisolone** is not feasible, the intravenous route can be used. High doses of GCs should not be used routinely.

Response criteria:

- Complete response (CR) — normalization of hemoglobin and absence of signs of hemolysis (normal bilirubin, LDH, haptoglobin, and reticulocytes), no need for transfusions.

- Partial response (PR) — an increase in hemoglobin of more than 20 g/L from baseline and absence of transfusions for more than 7 days, or normalization of hemoglobin with persistent signs of hemolysis.

- Non-response — failure to achieve response criteria.

- Steroid dependence — the need to take prednisolone** at a dose of more than 0.2 mg/kg/day to maintain response.

If there is no response to GC therapy within 3 weeks, the therapy is deemed ineffective and a switch to other lines of therapy is required.

CAD is a secondary disease associated with bacterial or viral infections in children; therefore, therapy is based on controlling the underlying disease. The need to prescribe drug treatment for CAD arises in transfusion-dependent patients. However, the use of GCs in such situations has a very limited effect (less than 15%).

Pneumocystis pneumonia is a potentially life-threatening complication in patients receiving high doses of GCs (more than 1 mg/kg/day) for more than one month.

It has been shown that a daily dose of prednisolone** ≥ 7.5 mg/kg/day for more than 3 months leads to an increased risk of fractures and the development of osteopenia. Adequate daily intake of vitamin D and calcium is indicated through diet, if possible, or in the form of dietary supplements, if necessary.

The number of upper gastrointestinal (GI) complications increases by 2.2–4.2 times in patients receiving GCs. Patients on long-term GC therapy require the prescription of antacid treatment (e.g., proton pump inhibitors).

Comments: upon achieving a sustained effect, the intervals between subsequent courses are increased to 7-14 days. This treatment option is well tolerated and causes fewer complications typical of GCs, but requires 6-8 repeated courses to achieve a sustained complete response.

Comments: #IVIG** can also be used as a therapeutic method if the patient has a negative direct Coombs test, but the medical history and other clinical and laboratory data point to AIHA. Due to its high cost and short-lived response to #IVIG**, this type of treatment is rarely recommended as monotherapy, most often as combination treatment with GCs.

Indication for the use of #IVIG**: severe hemolysis with a poor response to GCS; infants and young children; concomitant severe infectious conditions.

Comments: the plasmapheresis procedure in IgM-mediated AIHA (CAD/CBD) yields a better effect than in IgG-mediated AIHA.

Comments: When using GCS therapy prior to the initiation of #rituximab**, it should be continued until a response to #rituximab** is achieved, which is typically delayed. The minimum number of #rituximab** infusions required to assess a response is 2. The standard course consists of 4 weekly infusions. Maintenance therapy with #rituximab** is not indicated.

Repeated courses of #rituximab** are also effective upon relapse.

Therapy is generally well tolerated. The main adverse effects of #rituximab** include infusion-related reactions of varying severity, infections, progressive multifocal leukoencephalopathy, reactivation of viral hepatitis, and fulminant hepatitis. Untreated hepatitis B and C are contraindications to initiating #rituximab** therapy.

Vaccination should be postponed until B-cell recovery following therapy.

In patients with ALPS/ALPS-like syndromes requiring treatment, #rituximab** monotherapy is the preferred first-line treatment option.

Comments: #Mycophenolate mofetil** (#MMF**) is an immunosuppressive drug that reduces T- and B-cell proliferation by inhibiting inosine monophosphate dehydrogenase without a concomitant increase in the risk of infectious complications. This drug has been demonstrated to be safe and effective for the treatment of autoimmune cytopenias in children. #Mycophenolate mofetil** is highly effective in the treatment of steroid-resistant forms of AIHA. The drug is also effective in children with "warm" AIHA associated with ALPS or ALPS-like syndromes.

Treatment with #MMF** in children is initiated at a dose of 20–30 mg/kg/day divided into 2 oral doses; in adults, the initial dose of #MMF** is 1000 mg daily. The duration of therapy is at least 3 months to evaluate treatment efficacy. If a response is achieved, the recommended duration of therapy is 2 years, followed by a gradual taper over 6 months.

It is important to note that #MMF** typically has a delayed response, so its administration should be combined with a gradually tapering GCS dose over several weeks for initial control. #MMF** can also serve as maintenance therapy following a course of #rituximab** to reduce the need for repeated infusions.

The most common adverse effects of #MMF** include gastrointestinal disturbances (diarrhea, nausea, abdominal pain) and headache.

Comments: #Sirolimus is prescribed at a dose of 1–3 mg/day, after which the dose should be adjusted to maintain optimal serum trough concentrations of #sirolimus** at 4–15 ng/mL throughout the treatment period. Similar to #MMF**, #sirolimus** has a delayed response and can serve as maintenance therapy after a course of intensive therapy (high-dose GCS, #rituximab**).

The duration of therapy is at least 6 months; for responding patients, therapy is continued for 1.5–2 years with a subsequent gradual dose reduction over 6 months.

The most frequent adverse effects include mucositis, various infections, and elevated liver enzymes.

Comments: Cyclophosphamide** is used for many malignancies, but is also widely employed in the treatment of autoimmune diseases.

Low-dose oral regimens (1–2 mg/kg once daily) or high-dose intravenous regimens (400 mg/m² once every 3–4 weeks, 2–3 courses) exist. The use of high-dose cyclophosphamide** has been described mainly in adult patients; there is insufficient data on its use in the treatment of AIHA in children, and therefore it is considered as a third-line therapy in selected severe cases. The safety of cyclophosphamide** in children, as with any "DNA-toxic" drugs, remains a concern; however, cyclophosphamide** is still preferred over splenectomy for the treatment of patients who do not respond to #rituximab**.

Cyclophosphamide** has a more pronounced toxicity profile, especially when administered at high doses. Adverse events include hemorrhagic cystitis, myelosuppression, infertility, and the development of secondary malignancies. Patients receiving cyclophosphamide** therapy should have their blood counts monitored weekly and maintain adequate hydration on the days of administration (intravenous or oral).

3.2 Management of Cold Agglutinin Disease (Adults)

Cold agglutinin disease (cold AIHA, cAIHA) has significant pathogenetic differences from warm antibody AIHA, which dictates different approaches to the diagnosis and management of these conditions. Thus, cold AIHA, despite its name, is essentially a mature B-cell lymphoproliferative disorder with a low malignant potential. A characteristic clonal B-cell population is detected in virtually all patients with cAIHA. Frequently, cAIHA is characterized by monoclonal IgM paraprotein secretion. Unlike wAIHA, where autoantibodies are typically IgG, in cAIHA the antibodies belong to the class IgM, which causes significant pathogenetic differences in the mechanisms of hemolysis. Thus, IgM upon interaction with the antigen is able to trigger the classical complement activation pathway, resulting not only in the phagocytosis of opsonized erythrocytes, but also in intravascular, complement-mediated hemolysis. Thus, in cAIHA, there is a combination of intravascular and extravascular hemolysis. The predominant site of extravascular hemolysis in cAIHA is the liver, which determines the low efficacy of splenectomy in this group of patients. Glucocorticoid therapy is generally also of low efficacy. In this regard, therapies aimed at suppressing the pathological B-cell clone come to the forefront in cAIHA management.

Indications for initiating therapy in cAIHA are determined based on clinical manifestations and the degree of anemia. Thus, in the absence of anemia or in mild anemia without additional symptoms (acrocyanosis, Raynaud's syndrome, ulceronecrotic or trophic skin changes, thrombotic complications), a watch-and-wait strategy is permissible.

Comment: patients should be advised to avoid cold exposure and protect exposed areas of the body. Supportive care may include folic acid** and prophylactic anticoagulant therapy according to individual indications.

- #rituximab** at a dose of 375 mg/m² IV infusion once weekly (course of 4 weeks) [69].

- #rituximab** at a dose of 375 mg/m² on day 1, combined with #bendamustine** 90 mg/m² on days 1 and 2 (28-day cycle), for a total of 4 treatment cycles [70].

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

Comment: the choice of treatment regimen is made individually, based on the assessment of comorbidities, general somatic status, and patient age. The regimen including #bendamustine** has higher efficacy, but is more toxic and should therefore be considered in younger patients without severe comorbidities.

Objective parameters for evaluating the efficacy of ongoing therapy include hemoglobin levels, serum paraprotein concentration, and laboratory markers of hemolysis (LDH, bilirubin, reticulocytes). The cold antibody titer may remain high for a prolonged period and does not reflect the efficacy of the therapy administered. It is advisable to evaluate efficacy no earlier than 6–12 weeks after the completion of #rituximab** therapy due to its delayed action. In the absence of an effect, it is advisable to proceed to second-line therapy.

- #rituximab** at a dose of 375 mg/m² on day 1, in combination with #bendamustine** 90 mg/m² on days 1, 2 (28-day cycle), for a total of 4 treatment cycles (if #rituximab** monotherapy was used as the first line) [70];

- #bortezomib** at a dose of 1.3 mg/m² subcutaneously on days 1, 4, 8, 11 (cycle — 21 days) [71];

- #ibrutinib** 420 mg orally once daily [72];

- #daratumumab** 16 mg/kg once weekly (8 infusions), then 16 mg/kg once every 2 weeks (8 infusions), then 16 mg/kg once a month [73, 74].

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

Comment: the choice of the second-line therapy regimen is made individually, based on the assessment of the patient's comorbidity and general somatic status. In the absence of an effect, switching to an alternative treatment regimen is possible.

Comment: if symptoms of microcirculation impairment are present (acrocyanosis, Raynaud's syndrome, ulcerative-necrotic or trophic skin changes, thrombotic complications), 2-4 sessions of therapeutic plasmapheresis may be performed to reduce the level of circulating cold hemagglutinins in the blood serum. The effect of plasmapheresis procedures is temporary, and therefore the next step requires systemic immunosuppressive therapy.

Comment: patients with CAD have an increased risk of thrombotic complications; therefore, prophylactic anticoagulant therapy should be considered in patients who are not planned to receive immunosuppressive therapy or in patients with additional risk factors for thrombosis (congenital thrombophilia, history of thrombosis, etc.). Standard regimens for anticoagulant prophylaxis in CAD have not been established. In patients at risk for bleeding, the risk-benefit ratio must be carefully weighed when deciding on anticoagulant therapy.

Surgical treatment

Comments: autoantibodies in wAIHA cause cell destruction primarily in the spleen as a result of the interaction between antibody-coated cells and phagocytes. Splenectomy has proven high but short-term efficacy in adult patients with wAIHA. Data on the use of splenectomy in pediatric patients are limited, and the evaluation of the efficacy of this type of therapy is not clearly defined in children. Whenever possible, splenectomy should not be performed in children under 5 years of age due to the higher risk of postsplenectomy sepsis caused mainly by Streptococcus pneumoniae and Haemophilus influenzae.

Patients scheduled for splenectomy must undergo preoperative immunization against Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae type b at least 14 days before elective splenectomy or after the 14th day following emergency splenectomy. In addition, children who have undergone splenectomy should receive antibiotic prophylaxis until the age of 5 years or for a minimum of 1 year after splenectomy, regardless of the age at the time of surgery and preoperative vaccination.

Splenectomy is not indicated for the treatment of intravascular hemolysis mediated by complement-activating antibodies, nor in extravascular hemolysis mediated by complement, since CD3b-opsonized erythrocytes are destroyed by liver macrophages in addition to the spleen.

3.4 Other treatment

Comments: replacement transfusion is performed using donor red blood cells matched for blood group and Rh factor phenotype, as well as for rare blood groups when possible to prevent additional alloimmunization.

It is necessary to analyze the patient's blood samples for timely and complete determination of the red blood cell phenotype and detection of possible alloantibodies masked by autoantibodies. Careful typing for erythrocyte antigens is advisable in order to improve transfusion efficacy.

The dose of donor red blood cells is selected individually; leukoreduction and, in rare cases, washing are performed. Washing of red blood cells is indicated only in PCH to remove residual amounts of plasma containing complement.

In emergency cases when blood typing is impossible due to vital indications, the recipient is transfused with O (I) Rh-negative red blood cell-containing components in an amount of no more than 500 ml, regardless of the recipient's blood group and Rh affiliation.

Transfusion should be performed slowly and only in the amount necessary to improve symptoms of hypoxemia (approximately 3-5 ml/kg per transfusion) to minimize complications associated with the risk of alloimmune hemolysis.

If PCH, CAD/CAS is suspected, a blood component transfusion warmer must be used.

Blood transfusion therapy may be performed at higher hemoglobin levels in patients in the intensive care unit with known heart or kidney diseases.

Comments: folates, vitamin B12, and iron are necessary for red blood cell production, and the need for them increases as red blood cell production rises during hemolysis. Hyperferritinemia can be observed in active extravascular hemolysis, whereas intravascular hemolysis can lead to iron loss through the kidneys. There are reports of folic acid deficiency in patients with chronic hemolytic anemia of various etiologies. Additional intake of vitamins, in particular #folic acid**, is well tolerated and widely practiced.

4. Medical Rehabilitation and Sanatorium-Resort Treatment

• Social rehabilitation work and neuropsychological rehabilitation are recommended for pediatric patients with AIHA in order to improve quality of life and the possibility of social adaptation in society [42].

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

Comments:

- Ability to attend organized groups – patients in remission receiving maintenance therapy with MMF have no restrictions on attending children's groups.

- Ability to engage in physical activity and sports – patients with a complete or partial response to therapy, patients in remission during the GCS dose reduction stage, or on maintenance therapy with #MMF** or #sirolimus** have no contraindications for physical activity. Moreover, physical activity in such patients is associated with a lower risk of complications and faster recovery.

5. Prevention and Dispensary Observation

• For adult patients with AIHA, it is recommended to undergo routine follow-up visits (examinations, consultations) by a hematologist to monitor treatment efficacy and assess disease status once every 6 months. During the dose reduction and discontinuation stage of immunosuppressants, or in the presence of treatment toxicity, more frequently [3-6].

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

Comments: if necessary, the study is performed more frequently (in case of an infectious disease, deterioration of general condition).

Comments:

• Electrocardiography – once a year.

• Comprehensive abdominal ultrasound, renal ultrasound, echocardiography – once a year.

• X-ray densitometry – once a year, for patients receiving GCS.

• Targeted chest X-ray, chest computed tomography, abdominal MRI – as clinically indicated.

Comments:

• follow-up examination (examination, consultation) by a general dentist (or follow-up examination (examination, consultation) by a pediatric dentist) as clinically indicated;

• follow-up examination (examination, consultation) by an endocrinologist – once a year or as clinically indicated;

• follow-up examination (examination, consultation) by an ophthalmologist – once a year or as clinically indicated;

• follow-up examination (examination, consultation) by a gastroenterologist as clinically indicated;

In the presence of comorbidities, more frequent consultations by specialists and consultations by other specialists may be performed, depending on clinical indications.

Comments: a contraindication to vaccination with live vaccines is an immunodeficiency state, immunosuppression, or malignant neoplasms. Whenever possible, the live vaccine should be replaced with an inactivated one. Children with secondary immunodeficiency associated with malignant diseases and (or) immunosuppression are vaccinated with live vaccines after remission has been achieved, no earlier than 3 months after the end of immunosuppressive therapy. Inactivated vaccines are administered to children with all forms of immunodeficiency as usual; it is advisable to assess their immune response and administer an additional dose of the vaccine if the response is weak.

The GCS dose that causes immunosuppression is prednisolone** 2 mg/kg/day (or 20 mg/day for children weighing more than 10 kg) taken for 14 days or more; administration of live vaccines to these children is permitted 1 month or more after the end of therapy.

Vaccination of children who have received blood products with live viral vaccines is carried out at the intervals specified in Table No. 4.

Table No. 4. Intervals for administration of live vaccines after blood component or blood product transfusion [44]

6. Organization of Medical Care

Indications for planned hospitalization:

1) Evaluation to identify the primary/underlying disease;

2) Selection of therapy in case of insufficient efficacy of previously administered treatment;

3) Dynamic monitoring of disease status and assessment of therapy tolerability, with instrumental diagnostic methods performed as clinically indicated.

Indications for emergency hospitalization:

1) Severe anemia and/or other cytopenia;

2) Prolonged febrile fever during immunosuppressive therapy;

3) Suspicion of oncohematological pathology.

Indications for patient discharge from the hospital:

1) Stabilization of condition;

2) Completion of planned examination;

3) Selection of therapy and good treatment tolerability.

7. Additional Information

Since complex cases of "warm" autoimmune hemolytic anemia (AIHA) in children are most commonly associated with immune dysregulation syndromes (ALPS, ALPS-like disorders, and other congenital immune defects), #MMF** and #sirolimus become the therapy of choice for long-term management in patients with relapsing and steroid-dependent forms of the disease [37, 45, 46]. Therefore, prior to initiating GCS and normal human immunoglobulin** therapy, it is important to screen for underlying immunological disorders by assessing immunoglobulin levels, lymphocyte subpopulations, and screening for ALPS [1]. Children with alterations indicative of immune dysregulation and the development of Evans syndrome, even if a genetic immune defect has not been identified, carry a higher risk of poor treatment response and relapse. Such patients require closer monitoring [3].

Patients who have failed third- and fourth-line treatment strategies may require hematopoietic stem cell transplantation (HSCT); however, such escalation of therapy is rarely required for "warm" AIHA in pediatric practice, except as a radical treatment for the underlying disease in patients with congenital immune defects [1, 4].

Autoimmune lymphoproliferative syndrome (ALPS) is a genetic disorder causing defective lymphocyte apoptosis, characterized by chronic non-malignant lymphoproliferation, a predisposition to autoimmune diseases, and an increased risk of malignancy. Abnormal TCRαβ+CD4-CD8- T lymphocytes (double-negative T cells) are a hallmark of the disease, and their detection in peripheral blood is essential for establishing the diagnosis [47]. Chronic, relapsing, multilineage cytopenia is the most common autoimmune manifestation of ALPS and the most frequent indication for treatment. Immune cytopenia associated with ALPS is chronic; therefore, the early consideration of immunosuppressants other than GCS is warranted [47]. #MMF** and #sirolimus are the most studied drugs in ALPS with good efficacy [48]. #Rituximab** therapy and splenectomy should be avoided in ALPS. #Rituximab** is less effective in ALPS-associated AIHA compared to primary AIHA and is more toxic, with the development of prolonged hypogammaglobulinemia reported due to irreversible B-cell depletion [49].

Common variable immunodeficiency (CVID) represents a heterogeneous group of disorders characterized by hypogammaglobulinemia, low specific antibody titers, an increased risk of infections, and a predisposition to both autoimmune diseases and malignancies. Notably, autoimmune complications are diagnosed prior to the establishment of CVID in 49% of patients [50]. Classical CVID is considered a primary B-cell disorder; however, combined cases also exist. #Rituximab** provides a sustained response in 59% of patients with refractory cytopenia, while carrying a risk of severe bacterial infections. However, 24% of patients treated with #rituximab** developed severe infections, although the majority of them had previously undergone splenectomy or had not received IVIG replacement therapy [51]. Patients with CVID or other concomitant immunodeficiencies in whom #rituximab** therapy is being considered must be informed of the infectious risks, and immunoglobulin levels should be regularly monitored to ensure timely IVIG replacement therapy.

With a better understanding of the underlying immune defects associated with the development of "warm" AIHA, new targeted therapies are emerging [52–55]. For instance, mutations in the CTLA4 gene lead to a complex immune dysregulation syndrome with a high propensity for autoimmune cytopias, as well as autoimmune inflammatory lesions of the lungs, brain, and intestines [52]. Patients with LRBA gene mutations exhibit a lymphoproliferative syndrome, humoral immunity defects, and autoaggression, accompanied by a loss of CTLA4 function [53]. These patients often suffer from a chronic relapsing course of AIHA and Evans syndrome. Further research into novel therapeutic approaches using targeted drugs will be required for refractory "warm" AIHA in children [1].

Systemic lupus erythematosus (SLE) underlies 4–10% of warm AIHA cases [56, 57]. A positive direct antiglobulin test is part of the diagnostic criteria for SLE, but it does not always cause hemolytic anemia or require specific AIHA treatment. When necessary, systemic corticosteroids are the preferred initial therapy for SLE-associated AIHA. Despite a good initial response, approximately one third of patients experience a relapse [57], which is why higher doses or prolonged courses of corticosteroids are usually required to treat SLE-associated AIHA.

All second-line therapies used for primary AIHA are also used in SLE. #Rituximab** has been effective in treating refractory AIHA in children with SLE [58]. Despite the risk of prolonged B-cell depletion, the probability of infectious complications was low [58]. According to a few case descriptions, therapy with #sirolimus and #MMF** for refractory forms of autoimmune cytopenia associated with SLE is also effective [59, 60, 61].

Warm AIHA in children associated with giant cell hepatitis (GCH) deserves special mention. This combination is a rare, distinct condition that typically manifests in infancy [62]. Initial clinical and laboratory findings include jaundice, hepatosplenomegaly, hemolytic anemia, and a positive direct antiglobulin test. Signs of liver disease, such as cholestasis and elevated transaminase activity (ALT/AST), may follow the symptoms of hemolysis over a period ranging from several days to several months [63]. Liver ultrasound shows no specific abnormalities, and serological tests for viral, metabolic, and common autoimmune causes of hepatitis are negative [64]. The diagnosis is confirmed histologically by liver biopsy, which reveals marked alterations in liver architecture, diffuse giant cell transformation, and central-portal bridging fibrosis [64].

GCH with AIHA has a poor prognosis, culminating in fulminant hepatitis without aggressive treatment [62]. The management of such patients is challenging, and early initiation of treatment is critical to prevent irreversible liver failure [64]. Furthermore, the risk of severe and fatal infections is elevated in these cases [65]. In GCH with AIHA, supplementary prolonged immunosuppressive therapy with azathioprine is used [64]. Given the severity of GCH in AIHA, even despite its low incidence, liver function tests should be performed regularly in every child diagnosed with AIHA [4].

Cold agglutinin-mediated AIHA in children is usually short-lived and self-limiting. Management is primarily supportive, including adequate hydration and diuresis [23]. Treatment of the underlying infection, whenever possible, is one of the main goals in managing a patient with CAD. Corticosteroids are ineffective, other immunosuppressants are not routinely indicated, and splenectomy is ineffective because hemolysis occurs predominantly intravascularly.

In mixed-type AIHA, the management and treatment tactics for warm AIHA should be followed [1].

PCH has a good prognosis upon achieving remission; however, it often presents with severe and rapidly progressive anemia that can be life-threatening. This requires supportive care, including body temperature control, rest, adequate hydration, and avoidance of cold exposure [16, 66]. The decision to administer therapy is made on an individual basis depending on the clinical picture, complete blood count parameters, and the rate of anemia progression. PCH generally does not require treatment, but the administration of corticosteroids using the regimen for warm AIHA is acceptable [1].

Any primary condition contributing to the development of AIHA requires appropriate treatment in addition to AIHA management [1].

Criteria for Evaluating the Quality of Medical Care

References

Appendix A1. Working Group Composition

1. Zhanna A. Kuzminova – Candidate of Medical Sciences, Senior Researcher, Department of Optimization of Treatment for Hematological Diseases (Myeloproliferation and Anemia Study Group), Institute of Hematology, Immunology and Cell Technologies, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health; Hematologist, Consultative Department, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health; Member of the Russian Society of Pediatric Oncologists and Hematologists.

2. Alexey A. Maschan – Doctor of Medical Sciences, Professor, Corresponding Member of the Russian Academy of Sciences, Deputy General Director, Director of the Institute of Hematology, Immunology and Cell Technologies, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health; Member of the National Hematological Society, Member of the Russian Society of Pediatric Oncologists and Hematologists.

3. Natalia S. Smetanina – Doctor of Medical Sciences, Professor, Deputy Director of the Institute of Hematology, Immunology and Cell Technologies, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health; Member of the National Hematological Society.

4. Pavel E. Trahtman – Doctor of Medical Sciences, Associate Professor, Head of the Department of Transfusion Medicine, Collection and Processing of HSC, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health; Member of the Russian Society of Pediatric Oncologists and Hematologists, Member of the Russian Association of Transfusiologists.

5. Andrey B. Abrosimov – Candidate of Medical Sciences, Senior Researcher, Laboratory of Pharmacology and Healthcare Technology Assessment, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Russian Ministry of Health.

6. Elena A. Lukina – Doctor of Medical Sciences, Professor, Head of the Department of Hematology and Chemotherapy of Orphan Diseases, National Medical Research Center for Hematology of the Russian Ministry of Health.

7. Nina V. Tsvetaeva – Candidate of Medical Sciences, Hematologist, Department of Hematology and Chemotherapy of Orphan Diseases, National Medical Research Center for Hematology of the Russian Ministry of Health;

8. Rodion V. Ponomarev – Candidate of Medical Sciences, Hematologist, Department of Hematology and Chemotherapy of Orphan Diseases, National Medical Research Center for Hematology of the Russian Ministry of Health.

9. Latyshev Vitaly Dmitrievich – hematologist, Head of the Day Hospital of Hematology for Patients with Hereditary Metabolic Diseases at the National Medical Research Center for Hematology of the Russian Ministry of Health.

All members of the Working Group have confirmed no financial support or conflict of interest.

Appendix A2. Methodology for Developing Clinical Guidelines

Target audience for these clinical guidelines:

1. hematologist

2. pediatric hematologist-oncologist

2. pediatrician

3. general practitioner (family physician)

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

Table 2. Scale for grading levels of evidence (LE) for preventive, therapeutic, and medical rehabilitation methods, including those based on natural therapeutic factors (preventive, therapeutic, and rehabilitation interventions)

Table 3. Scale for assessing grades of recommendation (GR) for prevention, diagnostic, treatment, and medical rehabilitation methods, including those based on natural therapeutic factors (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

No.

Appendix B. Physician Action Algorithms

Diagnostic algorithm for a patient with suspected AIHA

• Prednisolone**

• #Rituximab**

• Cyclophosphamide**

• #Sirolimus

• #MMF**

Appendix C. Patient Information

What is autoimmune hemolytic anemia?

Autoimmune hemolytic anemia (AIHA) is a condition in which the body's own immune system mistakenly produces antibodies that attack and destroy the patient's red blood cells. Red blood cells carry oxygen from the lungs to all organs and tissues of the body. Their accelerated destruction (hemolysis) leads to anemia—a decrease in red blood cell count and hemoglobin concentration in the blood.

What are the main symptoms of autoimmune hemolytic anemia?

The development of hemolytic anemia is accompanied by a number of symptoms that allow the physician to suspect the diagnosis, including:

• General weakness, rapid fatigue, decreased performance;

• Shortness of breath, palpitations (especially during physical exertion);

• Pallor of the skin and visible mucous membranes;

• Jaundice of the skin and sclera (yellowing of the eyes) due to the release of the bilirubin pigment upon red blood cell destruction;

• Dark urine ("dark beer" or tea color);

• Dizziness and tinnitus are possible.

Symptoms may develop either gradually or acutely in the case of a hemolytic crisis. In case of a sharp increase in weakness, shortness of breath, or the appearance of palpitations at rest, you should immediately seek medical attention.

How is autoimmune hemolytic anemia diagnosed?

The diagnosis of autoimmune hemolytic anemia is established by a hematologist based on:

1. Complete (clinical) blood count, which reveals anemia, an increased count of young red blood cells (reticulocytes), and other characteristic changes.

2. General clinical blood chemistry (elevated bilirubin, lactate dehydrogenase [LDH] levels).

3. Urinalysis (presence of urobilinogen).

4. Direct antiglobulin test (direct Coombs test)—a key test confirming the presence of antibodies on the surface of red blood cells. It may not be positive in all cases.

5. Additional studies to clarify the AIHA variant and search for a potential underlying cause, which may include a bone marrow examination (to rule out a lymphoproliferative disorder, systemic connective tissue disease, and other conditions).

What are the principles of treatment for autoimmune hemolytic anemia?

The main goal of AIHA treatment is to suppress the pathological immune response, inhibiting the production of anti-red blood cell antibodies to prevent red blood cell destruction.

In severe anemia, transfusions of red blood cell components may be performed, although finding compatible blood components in AIHA is challenging.

What is the prognosis for autoimmune hemolytic anemia?

The prognosis for AIHA with timely diagnosis and treatment is favorable. Modern drug therapy options for AIHA allow achieving cessation of hemolysis in the vast majority of patients. Due to the possibility of disease relapse in patients who have achieved AIHA remission, regular follow-up care by a hematologist is necessary.

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

No.