Autoimmune Hemolytic Anemia (AIHA) is a group of hemolytic anemias in which abnormal immune function leads to hyperfunction of B cells that produce antibodies to their own red blood cells, and the red blood cells adsorb autoantibodies and/or complement, resulting in accelerated destruction and shortened life span of red blood cells. Hemolytic anemia. Foreign data show that the annual incidence of AIHA is (0.8-3.0)/100,000. AIHA can be divided into warm antibody type (37°C, 60%-80%), cold antibody type (20°C, 20%-30%) and mixed warm and cold antibody type (about 5%) according to the optimal temperature of reaction between autoantibodies and erythrocytes. About 50% of warm antibody AIHA are secondary to hematopoietic and lymphoproliferative diseases such as chronic lymphocytic leukemia, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, Castleman’s disease, myelofibrosis, solid tumors, immune diseases, infections, drugs, primary immunodeficiency diseases, pregnancy, and allogeneic hematopoietic stem cell transplantation. Diagnostic criteria and typing (a) Diagnostic criteria. 1. Hemoglobin level reaches the standard of anemia. 2. Red blood cell autoantibodies are detected. 3. At least one of the following: reticulocyte percentage > 4% or absolute value > 120×109/L; conjugated bilirubin < 100 mg/L; total bilirubin ≥ 17.1μ 2 mol/L (mainly elevated non-conjugated bilirubin). (B) Typing. 1. According to the clear etiology or not, it is divided into two categories: secondary and primary. According to the optimal temperature required for autoantibodies to bind to erythrocytes, there are warm antibody type, cold antibody type [including cold agglutinin syndrome (CAS) and paroxysmal cold hemoglobinuria (PCH)] and mixed type. Based on the results of autoantibody testing of erythrocytes, there are autoantibody-positive and autoantibody-negative types. Autoantibody-negative AIHA is diagnosed when the clinical symptoms are consistent with hemolytic anemia, except for other hemolytic anemias, and immunosuppressive therapy is effective. (iii) Warm antibody autoimmune hemolytic anemia. 1. Clinical and laboratory manifestations of hemolytic anemia, such as malaise, pallor, jaundice, splenomegaly and other clinical signs and symptoms, as well as increased serum indirect bilirubin, increased serum lactate dehydrogenase, decreased conjugated globulin and increased reticulocyte absolute value. A positive direct anti-human globulin test (Coombs test) is usually IgG, IgG+C3, or occasionally IgA. If the Coombs test is negative (including IgG, IgM, C3), but the clinical presentation is consistent with the effectiveness of immunosuppressive therapy such as adrenocorticosteroids and other hemolytic anemias, AIHA with negative Coombs test can be considered. 4. Autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis or other diseases such as lymphatic system tumors (including chronic lymphoma) should be excluded. (d) Cold agglutinin syndrome. 1. Clinical and laboratory manifestations consistent with hemolytic anemia: cyanosis of the auricles, nasal tips and fingers in a cold environment, which disappears after warming, and signs of anemia or jaundice; laboratory tests reveal elevated bilirubin, and ferritinuria in recurrent cases. 2. Positive cold agglutinin test. 3. Direct Coombs test is almost always complement C3 type. (E) Paroxysmal cold hemoglobinuria. The clinical and laboratory manifestations of hemolytic anemia are consistent with the following: episodes of hemoglobinuria after exposure to cold, rapid progression of anemia during episodes, elevated bilirubin in laboratory tests, and ferric hemoglobinuria in recurrent episodes. 2. Positive cold and heat hemolysis test. 3. Direct Coombs test is positive for complement type C3. The clinical manifestations of AIHA are diverse, with great variability in the speed of onset, degree of hemolysis and course of the disease. The clinical manifestations of anemia and hemolysis are often present, and secondary AIHA is often accompanied by the clinical manifestations of the primary disease. (A) Typical symptoms. Signs of hemolysis may be present, such as weakness, anemia, jaundice, change in urine color, splenomegaly, etc. In case of hemolytic crisis, patients may have back pain, chills, high fever, syncope, hemoglobinuria, etc. 4 2. Pallor and jaundice are seen in about 1/3 of patients. Splenomegaly is present in more than half of the patients and is usually mild to moderately enlarged, moderately firm, and not painful. In primary AIHA cases, about 1/3 have mild hepatomegaly, moderate stiffness without pain, and marked enlargement is rare. Lymph nodes are enlarged in only 23% of patients with primary AIHA and 37% of patients with AIHA secondary to lymphatic reticular disease. (B) Concomitant symptoms. 1. Warm antibody AIHA: Mostly secondary to other diseases, often with signs of the primary disease on top of the typical signs of hemolysis, and may be accompanied by enlarged liver, spleen, jaundice, and enlarged lymph nodes. When the onset of the disease is rapid, there may be anemia, high fever, chills, back pain, vomiting, diarrhea, and headache, irritability and coma when the anemia is obvious. Cold antibody type AIHA: cold agglutinin syndrome: aggravated by cold, often manifested as terminal limb cyanosis, even frostbite, gangrene, Raynaud’s phenomenon may occur, improving with warmth. 3. Paroxysmal cold hemoglobinuria: The onset of hemoglobinuria occurs minutes or hours after cold stimulation. Acute attacks may include chills, high fever, generalized weakness, abdominal discomfort, back and lower extremity muscle pain, nausea, and vomiting for several hours or days. It may be accompanied by splenomegaly, hyperbilirubinemia, and recurrent episodes of ferritinuria. Patients with syphilis may have Raynaud’s phenomenon and may also have urticaria. (a) Peripheral blood cell analysis. The peripheral blood picture shows varying degrees of hemoglobin reduction, anemia is generally positive 5-cell and positive pigment anemia, the proportion and absolute value of reticulocytes are significantly increased, and fragments of red blood cells are easily seen in the blood smear, spherical red blood cells and nucleated red blood cells can be seen. The white blood cell count is normal or mildly elevated, and the platelets are normal or elevated. (b) Bone marrow cell morphology. The bone marrow picture is characteristically juvenile erythroid proliferative marrow picture with inverted granulocyte/red ratio and occasional mild megaloblastic erythrocyte changes. The bone marrow is hypoproliferative with a decrease in whole blood cells and reticulocytes in the presence of reoccurrence crisis. (iii) Biochemical examination. Elevated total serum bilirubin, mainly indirect bilirubin, elevated lactate dehydrogenase, and decreased conjugated pearl protein in acute hemolysis may be seen, but these indicators are nonspecific. (iv) Specific tests. 1. Erythrocyte autoantibody test. (1) Direct antiglobulin test (DAT) detects autoantibodies to the membrane of the covered red blood cells. The optimal binding temperature between warm autoantibodies and red blood cells is 37 ℃, while the optimal binding temperature between cold autoantibodies and red blood cells is 0~5 ℃. (2) Indirect antiglobulin test (IAT) detects free warm antibodies in the serum. (3) The cold agglutinin test detects cold agglutinin in the serum. Cold agglutinin is an IgM type cold antibody, and the best binding temperature with red blood cells is 0-5 ℃. CAS can be diagnosed when the cold agglutinin potency>1:32. The DAT result of CAS is positive for complement C3. 6 (4) Hot and cold hemolysis test for cold and hot diphasic hemolysin (D-L antibody). The D-L antibody is an IgG-type cold and heat hemolysin that binds to red blood cells at 0-4°C and adsorbs complement, but does not hemolysis; hemolysis occurs at 30-37°C. The cold and heat hemolysis test for PCH is positive, and the DAT result is positive for complement C3. 2. Etiological examination. The diagnosis is primary AIHA if there is no underlying disease and secondary AIHA if there is an underlying disease (Table 1). Table 1 Common causes of secondary autoimmune hemolytic anemia Lymphoproliferative disorders Chronic lymphocytic leukemia Other non-Hodgkin’s lymphoma Monoclonal IgM of undetermined significance Gammaglobulinemia Hodgkin’s lymphoma Autoimmune lymphoproliferative syndrome Solid tumor/ovarian dermatomal cyst Autoimmune diseases Systemic lupus erythematosus Hashimoto’s thyroiditis Ulcerative colitis Infections Mycoplasma infections EB Viral infections Cytomegalovirus infections Microvirus infections Human immunodeficiency virus infections Hepatitis virus infections Rotavirus and other enterovirus infections Adenovirus infections Respiratory syncytial virus and influenza virus infections Immunodeficiency Common variant immunodeficiency diseases Primary combined immunodeficiency diseases Drugs Purine analogues: fludarabine, cladribine Cephalosporins: cefotetan, ceftriaxone Piperacillin β-lactamase Inhibitors: tazobactam, sulbactam 7 Blood group incompatibility Allogeneic hematopoietic stem cell transplantation/solid organ transplantation Alloimmunization Chronic hemolysis after blood transfusion V. Differential diagnosis (a) Paroxysmal sleep hemoglobinuria. It is similar to paroxysmal cold hemoglobinuria, but the former occurs mostly after sleep and is not associated with cold stimuli, and can be differentiated from the latter by negative cold hemolysis test and positive acid hemolysis test and sugar water test. (b) Thrombotic thrombocytopenic purpura. It is a microangiopathic hemolysis with a negative anti-human globulin test and a large number of lytic cells in the blood smear in addition to small irregularly circumscribed spherical cells, which can be differentiated from warm antibody type AIHA. (iii) Hereditary spherocytosis. The main manifestations are anemia, jaundice, and splenomegaly, similar to AIHA, but the anti-human globulin test is negative and the autohemolytic test is enhanced, which is obviously corrected by adding glucose, while AIHA is mostly uncorrected. (iv) Other diseases that can cause Raynaud’s phenomenon. The former is not associated with cold and has a negative condensation set test. Treatment (a) Supportive treatment. The presence of autoantibodies in AIHA makes crossmatching more difficult and increases the risk of hemolytic transfusion reactions due to alloantibodies. The timing of transfusion should be determined by the degree of anemia, the presence of obvious symptoms, and the speed of occurrence. For patients with acute hemolytic anemia, those who can exclude homozygous antibodies at the onset of severe symptoms should be transfused with red blood cells immediately. For patients with chronic anemia, transfusion is not necessary if the hemoglobin is above 70 g/L; if the hemoglobin is between 50 and 70 g/L, transfusion may be appropriate if there are intolerable symptoms; if the hemoglobin is below 50-70 g/L, transfusion should be given. 3. Test the specificity of autoantibodies against ABO and Rh blood group, and perform the selection and cross-matching test on the donor. If the crossmatch is not completely compatible, use the least reactive of the multiple specimens for transfusion. Slowly titrate and closely observe for transfusion reactions. 4. Do not emphasize the use of washed red blood cells during resuscitation. Plasmapheresis or immunosuppressive therapy should be used if conventional treatment is not effective. The use of glucocorticoids before transfusion can reduce and mitigate the occurrence of transfusion reactions. In addition, attention should be paid to alkalinization and diuresis, bile removal, and electrolyte balance. (ii) Glucocorticoids. Glucocorticosteroids are recommended in the absence of contraindications to glucocorticosteroid use. The recommended starting dose is 1 mg/(kg-d) based on prednisone, which can be converted to intravenous infusion of dexamethasone and methylprednisolone as appropriate. Consider reducing the dose of glucocorticosteroids until the red cell volume is greater than 30% or the hemoglobin level is stable at 100 g/L or higher. If these results are not achieved after 4 weeks of treatment with the recommended dose, second-line dosing is recommended. Acute severe AIHA may require 100-200 mg/d of methylprednisolone for 10-14 days to control the disease. The dose is maintained for 1 month after normalization of hemoglobin and then tapered. The rate of reduction is determined at the discretion of the patient, and hemoglobin levels and absolute reticulocyte values are monitored closely during this period. Consider discontinuing glucocorticoids when the prednisone dose is reduced to 5 mg/d and remission continues for 2 to 3 months. Cold antibody AIHA is mostly secondary and the treatment is different from warm antibody AIHA, see secondary AIHA treatment. (iii) Second-line treatment. Second-line therapy is recommended for (1) glucocorticoid resistance or maintenance doses above 15 mg/d (based on prednisone); (2) other contraindications or intolerance to glucocorticoid therapy; (3) AIHA relapse; and (4) refractory/severe AIHA. Second-line therapy includes splenectomy, rituximab, cyclosporine A, and cytotoxic immunosuppressants. 1. Splenectomy. Splenectomy may be considered for refractory warm antibody AIHA, but there are no indicators to predict the efficacy of splenectomy. Other complications include venous thrombosis, pulmonary embolism, and pulmonary arterial hypertension. 2. Rituximab. 10 The dose of rituximab is 375 mg/(m 2-d) for 4 doses on days 1, 8, 15, and 22. Smaller doses of rituximab (100 mg/d) have also been reported to reduce the financial burden on patients and reduce adverse effects without decreasing efficacy. Monitoring of B-lymphocyte levels can guide the management of complications of rituximab including infection and progressive multifocal leukoencephalopathy. Patients with hepatitis B virus infection should be treated with rituximab when antivirals are effectively controlled and administered continuously. 3. Cytotoxic immunosuppressants. The most commonly used ones are cyclophosphamide, azathioprine, and vincristine, etc. The general efficiency is 40%-60%, and in most cases they are still used in combination with glucocorticoids. Adverse effects of cyclosporine A include gingival hyperplasia, hair growth, high blood pressure, increased bilirubin, and impaired renal function. Because cyclosporine A requires an effective blood level before it can be effective, it is recommended that it be used initially in combination with glucocorticoids. The use of tacrolimus and mycophenolate has also been reported in refractory AIHA. (iv) Treatment of secondary AIHA. Secondary AIHA requires aggressive treatment of the primary disease, and the rest of the treatment is the same as for primary AIHA. Most cold-antibody AIHA is secondary, and it is important to treat AIHA with concomitant insulation. (v) Other medications and treatments. Intravenous immunoglobulin is effective in some patients with AIHA. Plasma exchange is effective for IgM-type cold antibodies (80% of IgM-type antibodies are free at 37°C),11 but it is not effective for other warm antibodies adsorbed on red blood cells, and the exchange brings in a large amount of complement. VII. AIHA efficacy criteria (a) Cured. If the AIHA is secondary to the infection, it will be cured after the primary disease is cured. Patients with CAS have normal cold agglutinin potency and patients with PCH have negative cold and heat hemolysis tests. (ii) Complete remission. The patient has no clinical symptoms, normal erythrocyte count, hemoglobin level and reticulocyte percentage, and normal serum bilirubin level; DAT and IAT are negative. (iii) Partial remission. The clinical symptoms have basically disappeared, hemoglobin>80g/L, reticulocyte percentage<4%, total serum bilirubin<34.2μmol/L. DAT is negative, or still positive but the potency is significantly lower than before. (iv) Ineffective. (iv) Ineffective. There are still varying degrees of anemia and hemolytic symptoms, and laboratory tests do not meet the criteria for partial remission. 12 Annex: Autoimmune Hemolytic Anemia Diagnostic and Treatment Guidelines (2022 Edition) Validation Expert Group (in order of last name) Team Leader: Huang Xiaojun Members: Wang Jing, Fu Haixia, Xu Lanping, Jiang Qian, Jiang Hao, Zhang Xiaohui, Yang Shenmiao, Zhang Yuanyuan, Jia Jinsong, Huang Xiaojun, Lu Jin