In order to further improve the diagnosis and treatment of aplastic anemia (AA) in China, the Red Blood Cell Disease (Anemia) Group of the Hematology Branch of the Chinese Medical Association has reached the following expert consensus on the diagnosis and treatment of AA in China, based on extensive consultation with relevant experts in China and with reference to the British Committee for Standards in Hematology (BCSH) Guidelines for the Diagnosis and Treatment of AA.
I. Definition and pathogenesis of AA
AA is a bone marrow hematopoietic failure caused by multiple etiologies and pathogenesis, mainly manifested by low proliferation of bone marrow nucleated cells, reduction of whole blood cells, and anemia, bleeding and infection caused by them. It is believed that abnormal activation and hyperfunction of T lymphocytes causing bone marrow damage, hematopoietic cell apoptosis and hematopoietic failure play a major role in the pathogenesis of primary acquired AA. Congenital AA is rare, mainly Fanconi anemia (autosomal recessive), dyskeratosis congenita (DKC), Diamond-Blackfan anemia (DBA), and Shwachmann-Diamond syndrome (SDS). This guideline mainly discusses primary acquired AA.
II. Diagnostic recommendations for AA
1. Classification of bone marrow failure diseases: AA belongs to bone marrow failure (BMF) diseases. bMF can be divided into congenital and acquired, while acquired BMF is divided into primary and secondary.
(1) Primary BMF: Primary BMF mainly includes (1) BMF derived from hematopoietic stem cell mass abnormalities, such as paroxysmal sleep hemoglobinuria (PNH) and myelodysplastic syndrome (MDS); (2) autoimmune-mediated BMF, which in turn includes cellular immune-mediated BMF (e.g., AA) and autoantibody-mediated BMF; and (3) hematocrit of undetermined significance, which some British scholars have proposed that this condition is a transitional stage of a specific disease that can progress to MDS, other hematologic disorders, or a new disease.
(2) Secondary BMF: There are many factors that cause secondary BMF, including (1) bone marrow hypoproliferative hematopoietic system tumors, such as hairy cell leukemia (HCL), large granular lymphocytic leukemia (LGLL), multiple myeloma (MM), or BMF secondary to radiation or chemotherapy; (2) infiltration of non-hematopoietic system tumors; (3) myelofibrosis; (4) severe nutritional anemia; (5) physical, chemical, and biological factors that cause acute hematopoietic Acute hematopoietic arrest caused by chemical or biological factors.
2. Laboratory tests for the diagnosis of AA
(1) Required tests: ①Routine blood tests: white blood cell count (WBC) and classification, red blood cell count (RBC) and morphology, hemoglobin (Hb) level, reticulocyte percentage and absolute value, platelet count (BPC) and morphology. ②Multi-site bone marrow aspiration: including at least iliac bone and sternum. Bone marrow smear analysis: degree of hematopoietic cell proliferation; granulocyte, red and lymphatic lineage cell morphology and stage percentage; megakaryocyte count and morphology; small granulocyte hematopoietic cell area; presence of abnormal cells, etc. ③Bone marrow biopsy: at least 2 cm of bone marrow tissue (iliac bone) specimens were taken to assess the degree of bone marrow proliferation, the proportion of cells of each lineage, the distribution of hematopoietic tissue (with or without focal CD34+ cell distribution, etc.), and the presence of bone marrow infiltration, bone marrow fibrosis, etc. ④ Flow cytometry was performed to detect the number of bone marrow CD34+ cells. ⑤ Liver, kidney and thyroid function, blood biochemistry and virology (including hepatitis virus, EBV, CMV, etc.). ⑥Serum ferritin, folic acid and vitamin B12 levels. (vii) Flow cytometry to detect PNH clone. (viii) Autoantibodies and rheumatic antibodies. ⑨ Cytogenetics: routine karyotype analysis, fluorescence in situ hybridization (FISH) and screening for hereditary diseases (chromosome breakage test is recommended for children or those with family history). ⑩ Imaging tests (e.g. chest
x-ray or CT, abdominal ultrasound, etc.) and electrocardiogram.
(2) Optional tests: hospitals with conditions can carry out the following tests: ① bone marrow hematopoietic cell membrane autoantibody test; ② lymphocyte subpopulation test, such as T-cell subpopulation, CD4+ cell subpopulation, etc.; ③ hematopoietic regulatory factor test, such as IFN-ǐ, TNF-, IL-2, etc.
3. Diagnostic criteria for AA.
(1) Routine blood tests: decreased whole blood cells, corrected reticulocyte ratio
(3) Bone marrow biopsy (iliac bone): hypoplasia of whole sections, decreased hematopoietic tissue, increased adipose tissue and/or non-hematopoietic cells, no increase in reticulocyte sclerosis, and no abnormal cells.
(4) Exclusion tests: congenital and other acquired, secondary BMF disorders must be excluded.
4. Determination of the degree of AA (typing).
(1) Heavy from the diagnostic criteria (Camitta criteria): ①Bone marrow cell hyperplasia <25% of normal; if ≥25% of normal but <50%, the residual hematopoietic cells should be <30%. (2) Blood count: two of the following three items are required: ANC <0. 5X109/L; corrected reticulocytes <1% or absolute value <20X109/L; BPC <20X109/L. (3) If ANC <0.2×109/L is very heavy AA.
(2) Diagnostic criteria for non-heavy AA: AA that does not meet heavy criteria.
Three, AA treatment recommendations
1. Supportive therapy.
(1) Component blood transfusion: the indication for blood transfusion is generally Hb<60g/L. The threshold for blood transfusion (Hb≤80g/L) can be relaxed in old age (≥60 years), low compensatory response capacity (such as with heart and lung disorders), increased oxygen demand (such as infection, fever, pain, etc.), and aggravated lack of oxygen supply (such as blood loss, pneumonia, etc.), and try to transfuse red blood cell suspension. Irradiated or filtered erythrocytes and platelet suspension should be transfused for allogeneic hematopoietic cell transplantation. Those with risk factors for platelet depletion [infection, bleeding, antibiotics or anti-thymocyte globulin/anti-lymphocyte globulin (ATG/ALG), etc.] or heavy disease should receive a prophylactic platelet transfusion threshold of <20X109/L and <10X109/L for stable disease. those with severe bleeding are not subject to these criteria and should be actively transfused with a single concentrated platelet suspension to The platelet count should be brought up to a relatively high level. Those with ineffective transfusions due to the development of anti-platelet antibodies should be transfused with HLA-matched platelets. Granulocyte transfusion may be considered in cases of severe infection with life-threatening granulocyte deficiency when the combination of antibiotics and G-CSF is ineffective.
(2) Other protective measures: Patients with heavy AA should be placed in protective isolation, or in a laminar flow ward if available; avoid bleeding, prevent trauma and strenuous activity; eliminate exposure to risk factors, including drugs that damage bone marrow and inhibit platelet function; and provide the necessary psychological care. Dietary hygiene needs to be observed and antifungal drugs can be applied prophylactically. Prophylactic antiviral therapy, such as acyclovir, is recommended for transplantation and ATG/ALG treatment. Pneumocystis carinii infection needs to be prevented after bone marrow transplantation, such as with cotrimoxazole (SMZco), but it is not necessary to apply it routinely for those treated with ATG/ALG.
(3) Treatment of infection: AA patients with fever should be treated according to the treatment principle of “neutropenia with fever”. The use of initial antibiotics should follow the principle of “heavy hitting”, and after the bacteriological basis, the targeted antibiotics should be selected according to the drug sensitivity (“descending ladder” selection). Antifungal therapy should be given to those who are ineffective in antibacterial therapy or who have fever again after initially being effective. Timely immunosuppressive therapy (IST) is an effective means of reducing infection from the patient. Effective antibiotics supplemented with G-CSF and granulocyte infusion can improve anti-infective efficacy.
(4) Iron removal therapy: Patients should be given iron removal therapy if their serum ferritin level exceeds 1000ug/L due to long-term blood transfusion.
(5) Vaccination: There have been some reports suggesting that vaccination can lead to recurrence of BMF or AA, so vaccination is not recommended unless absolutely necessary.
2. Treatment of AA as a disease
Once AA is diagnosed, the severity of the disease should be clarified and treated as soon as possible. The standard treatment for severe AA is IST with ATG/ALG and cyclosporine (CsA) plus hematopoietic therapy for patients ≥40 years of age or <40 years of age without HLA compatible sibling donor; for patients <40 years of age with severe AA with HLA compatible sibling donor, HLA compatible sibling donor bone marrow transplantation is preferred if there is no active infection or bleeding. Donor bone marrow transplantation should only be used in young patients with severe AA who have failed ATG/ALG and CsA therapy. Bleeding and infection must be controlled prior to bone marrow transplantation. Peripheral blood stem cell transplantation with G-CSF mobilization is not recommended. Transfusion-dependent non-heavy AA can be treated with CsA + hematopoietic (androgens, hematopoietic growth factors) and treated as heavy AA if treatment is ineffective for 6 months. Non-heavy AA not dependent on blood transfusion can be treated with CsA and/or hematopoietic treatment.
(1) IST plus hematopoietic treatment: ①ATG/ALG combined with CsA for IST: heavy AA; transfusion-dependent non-heavy AA and CsA combined with hematopoietic treatment for 6 months is ineffective. ②ATG/ALG: rabbit-derived ATG/ALG (French, German) at a dose of 3-5 mg?kg-1?d-1, pig-derived ALG (Chinese) at a dose of 20-30 mg?kg-1?d-1. ATG/ALG should be administered for 5 d and infused intravenously for 12-18 h daily. 1/10 of the single ATG/ALG (rabbit-derived ATG 2.5 mg from France, rabbit-derived ALG 2.5 mg from Germany) should be administered first. If a serious systemic reaction or allergic reaction occurs, stop ATG/ALG infusion and promptly administer anti-allergic treatment, at the same time, determine the positive intravenous test of ATG/ALG and disable ATG/ALG; if the intravenous test is negative, administer regular ATG/ALG. If the intravenous test is negative, regular ATG/ALG treatment will be administered. Apply adrenal glucocorticosteroids simultaneously with daily ATG/ALG to prevent allergic reactions. Total daily glucocorticoids are converted to methylprednisolone or dexamethasone or hydrocortisone at 1 mg?kg-1?d-1 of prednisone and infused via a separate intravenous route in conjunction with ATG/ALG. Acute phase adverse reactions include hypersensitivity reactions, fever, rigors, rash, hypertension or hypotension, and fluid retention. Patients should have a tracheotomy kit and epinephrine at their bedside. Maintain BPC >10×109/L during dosing. Because of the anti-platelet activity of ATG/ALG, platelet suspension should not be infused with ATG/ALG infusion. Serum sickness reaction (arthralgia, myalgia, rash, mild proteinuria and thrombocytopenia) usually appears about 1 week after ATG/ALG treatment, so glucocorticoids should be used in full dose until 15d, and then the dose should be reduced, usually after 2 weeks (total course of treatment is 4 weeks). For those who develop serum sickness, intravenous adrenal glucocorticoid shock therapy should be applied, with the total daily amount of prednisone 1 mg?kg-1?d-1 converted to hydrocortisone or methylprednisolone, and the dosage and duration of treatment should be adjusted according to the patient’s condition. The second ATG/ALG treatment was recommended for patients who had failed the first ATG/ALG treatment or relapsed. The interval between the two treatments is 6 months, as most patients do not show efficacy until about 6 months. For the 2nd course of ATG/ALG, another animal species source of ATG/ALG is chosen to reduce the risk of allergic reactions and severe seropathy. CsA: CsA is available in three dosage forms: injection, oral solution, and capsule, and the latter two are mostly used from treatment. The exact effective blood concentration of CsA in the treatment of AA is not clear, and the effective blood concentration window is large, generally the target blood concentration (trough concentration) is 150-250ug/L in adults and 100-150ug/L in children. The main adverse effects of CsA are gastrointestinal symptoms, gingival hyperpigmentation, pigmentation, muscle tremor, liver and kidney impairment, rarely headache and blood pressure changes, most patients have mild symptoms or symptomatic treatment to reduce, if necessary, reduce the dose or even discontinue. Blood pressure, liver and kidney function should be tested regularly while taking CsA. ④IST in elderly patients: ATG treatment is never age-restricted, but comorbidities should be evaluated before treatment in elderly AA patients. the risk of bleeding, infection and cardiovascular events is greater in elderly AA patients treated with ATG/ALG compared to younger patients. Therefore, attention needs to be paid to cardiac function, liver function, lipids, and glucose tolerance in elderly patients. In view of the risk of nephrotoxicity and hypertension, CsA therapy in elderly AA patients is recommended to have a trough blood concentration of 100-150ug/L.5 Pro-hematopoietic therapy: Androgens can stimulate bone marrow erythropoiesis and reduce excessive menstrual bleeding in female patients, and are the basic pro-hematopoietic drugs for AA therapy. In combination with CsA, it is effective in the treatment of non-heavy AA. Stanozolol (2mg, 3 times daily) or testosterone undecanoate (40mg, 3 times daily) is generally applied, and liver function should be reviewed regularly. GM-CSF and G-CSF have been reported to exert a pro-hematopoietic effect when used in combination with immunosuppressive agents. The duration of hematopoietic factors should be determined by the patient’s blood and bone marrow response. Generally, it should not be too short. It can be used 3 times a week for 1 month, 2 times a week for 1 month, or 1 time a week for 1 month, and the total course of treatment is usually not less than 3 months. (6) Follow-up: Patients treated with ATG/ALC and CsA should be closely followed up with regular examinations for timely evaluation of efficacy and adverse effects (including evolution to clonal diseases such as PNH, IVIDS and AML, etc.). The recommended follow-up observation points are 3 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 5 years, and lO years after ATG/ALG administration.
(2) HLA-matched sibling donor bone marrow transplantation: ①Applicable conditions: heavy or very heavy aa patients aged <40 years with hla-matched sibling donors. Heavy aa patients over 40 years of age can also be treated with hla-identical sibling donor bone marrow transplantation after atg/alg combined with csa therapy has failed. ②Stem cell quantity: at least 3×108/kg for single nucleated cells and at least 3×106/kg for CD34+ cells are recommended for return transfusion. ③Graft pretreatment and post-transplant IST: for patients aged <30 years, the standard pretreatment regimen is cyclophosphamide 50 mg?kg-1?d-1X4d (-5 to -2d) and rabbit-derived ATG 3.75 mg?kg-1?d-1×3d (- 5～-3d), and methylprednisolone 2mg?kg-1?d-1×3d(-5～-3d). Methylprednisolone is not usually used in pediatric bone marrow transplant patients. The recommended post-transplant 1ST is CsA5mg?kg-1?d-1 orally in 2 doses, starting from the 1st 1d and decreasing from the 9th month for 12 months to prevent late graft failure; ② short term application of methotrexate 15Mg/m2,+ld, 10mg/m2+3,+6,+lld. There is no optimal pretreatment regimen for patients aged >30 years.
(3) Bone marrow transplantation from HLA-compatible unrelated donors: ①Applicable conditions: HLA-completely compatible (class I antigens and class II antigens at the DNA level) donors; age <50 years (50-60 years, must be in good general condition); heavy or very heavy AA patients; no HLA-compatible sibling donors; failure of at least one ATG/ALG and CsA treatment; no active infection and no bleeding at the time of bone marrow transplantation. bleeding at the time of bone marrow transplantation. ②Pre-treatment: cyclophosphamide 300mg?m-2?d-1X4d; fludarabine 30mg?m-2?d-1X4d; rabbit-derived ATG 3.75mg?m-2?d-1X4d (or alemtuzumab 0.2mg?kg-1?d-1 to a maximum dose of 10mg/d x 5d); CsA 1mg?kg-1?d-1,-6 ~-2d, 2mg?kg-1?d-1,-1 to +20d, followed by 8mg?kg-1?d-1 orally; (3) if ATG is used instead of alemtuzumab, methotrexate 10mg/m2,+1d, 8mg/m2+3d, +6d. In elderly patients, ATG dosage is reduced and 200cCy total body irradiation is added. Current bone marrow transplantation for severe AA recommends avoiding irradiation-containing pretreatment regimens in pediatric and younger patients, even at low doses, and replacing them with fludarabine. Low-dose irradiation given to older patients may be beneficial in reducing rejection.
(4) Other immunosuppressive agents: ① High-dose cyclophosphamide: Due to the high lethality and serious adverse effects of high-dose cyclophosphamide (45 mg?kg-1?d-1X4d), it is not recommended for primary patients without bone marrow transplantation or AA patients who have failed ATG combined with CsA therapy. ②Mycophenolate mofetil (MMF): Research on this drug has focused on the treatment of refractory AA, but multiple center studies have shown that MMF is ineffective in refractory AA. ③Pulcoflora (FK506): It has the same signaling pathway as CsA to inhibit T-cell activation but has stronger effects, less nephrotoxicity, and does not cause gingival hyperplasia, so it has been used to replace CsA for the treatment of AA, and the preliminary results are encouraging and worthy of clinical exploration. ④Rapamycin: It has synergistic effect with CsA in suppressing T-cell immunity, but the latest findings show that adding rapamycin to ATG+CsA does not improve the response rate of patients to treatment. Clinical studies of rapamycin+CsA for refractory, relapsed AA are ongoing. ⑤ Anti-CD52 monoclonal antibody: clinical trials are underway.
3. Treatment of patients with abnormal clones of AA: A small number of AA patients have cytogenetic clonal abnormalities at diagnosis, commonly +8, +6, 5q- and chromosome 7 and 13 abnormalities. Usually the abnormal clones represent only a very small fraction of the total split image, may be transient and can disappear on their own. Several studies have shown that AA patients with and without the above genetic abnormalities respond similarly to IST. Patients with abnormal karyotypes should undergo a bone marrow cytogenetic analysis every 3-6 months, and increased abnormal schizograms suggest disease transformation.
4. Management of AA patients with significant PNH karyorrhexis: A small number of PNH clones can be detected in AA patients, who have reduced bone marrow cells but do not appear hemolyzed. Usually only monocytes and neutrophils are involved alone and in a small percentage. The recommended management for these patients is the same as for AA patients without PNH clones. Patients with AA with significant PNH clones (>50%) should be treated with ATG/ALG with caution and should be treated as PNH for the time being.
The treatment of pregnant AA patients: AA can occur during pregnancy, and some patients need supportive therapy. after pregnancy in AA patients, the disease may progress. The main treatment for pregnant AA patients is to give supportive therapy and platelet transfusion to maintain the patient’s platelet count above 20X109/L. ATG/ALG is not recommended during pregnancy and CsA therapy may be given. The patient’s pregnancy status, blood count and vital organ function should be closely monitored during pregnancy.
IV. Efficacy criteria of AA
1. Basic cure: anemia and bleeding symptoms disappear, Hb reaches 120g/L for men and 110g/L for women, WBC reaches 4×109/L, BPC reaches 100×109/L, and no recurrence for more than 1 year of follow-up.
2. Remission: anemia and bleeding symptoms disappeared, Hb reached 120g/L for men and 100g/L for women, WBC reached about 3.5X109/L, BPC also increased to some extent, and the disease was stable or continued to progress in 3 months of follow-up.
3. Obvious progress: anemia and bleeding symptoms improved significantly without blood transfusion, and Hb increased by more than 30g/L compared with the common value in 1 month before treatment, and could be maintained for 3 months.
Those who judge the above three efficacy criteria should not be transfused within 3 months.
4. Ineffective: After adequate treatment, the symptoms and blood routine do not reach significant progress.
The above “Expert Consensus on the Diagnosis and Treatment of AA” was jointly discussed and collectively revised by the experts of the Red Blood Cell Disease Group, and there are inevitably defects and inappropriate points in the text.