Aplastic anemia (AA) is a group of bone marrow failure syndromes characterized by peripheral blood allogeneic cytopenia, reduced bone marrow hematopoietic cell proliferation without abnormal cell infiltration and fibrous tissue hyperplasia. Depending on the etiology, retrolithiasis can be classified as congenital or acquired, and the latter can be further classified as primary or secondary depending on the presence or absence of a clear causative factor.
Acquired remitting disorders account for the majority of cases, often without a clear cause, and are primary. Unless otherwise specified, the term “reocclusion” usually refers to acquired reocclusion. A clear diagnosis, precise etiology and severity typing of reoccurrence are of great value in the development of treatment measures and prognostic assessment of patients.
Clinically, all patients presenting with reoccurrence should be carefully evaluated using appropriate diagnostic measures with the following objectives: (1) to confirm the diagnosis of acquired reoccurrence, except for other disorders manifesting as bone marrow hypoproliferation and complete blood cytopenia; (2) to perform severity staging of reoccurrence; (3) to determine whether abnormal cytogenetic clones or PNH clones are present; and (4) to exclude late onset congenital bone marrow hematopoietic failure.
I. Diagnostic and severity typing criteria for remitting disease: remitting disease lacks specific clinical and laboratory examination manifestations. In a sense, the diagnostic criteria for remitting disease can in fact be interpreted as exclusion criteria for all other known bone marrow hematopoietic failures. The International Study Group on Granulocytopenia and Aplastic Anemia (1987) proposed that the diagnosis of aplastic anemia [1] must meet at least 2 of the following 3 points hemoglobin <100 g/L and platelets <50x109/L; (3) neutrophils <1.5x109/L.
If a patient has a decrease in peripheral blood secondary or tertiary blood cells that does not meet the above criteria, the patient should not be diagnosed with reocclusion, but changes in blood counts should be monitored closely. After the diagnosis of reocclusion, the clinical type should be further determined. At present, the international standard of Camitta (1976) is commonly used to classify reocclusion into heavy (SAA) and non-heavy (NSAA), and the diagnostic criteria of very heavy reocclusion (VSAA) were added in 1988.
In 1987, the Fourth National Conference on Recurrent Disorders formulated the diagnostic criteria for recurrent disorders in China, which have been used since then. Compared with the international diagnostic staging criteria for reoccurrence, our criteria not only emphasize blood and bone marrow examination, but also incorporate clinical manifestations into the aplastic anemia staging criteria, and staging them into acute aplastic anemia and chronic aplastic anemia. There is a high degree of agreement between domestic typing and Camitta typing, with the latter emphasizing the severity of hematopoietic failure and the former emphasizing the rapidity of the development of this failure in addition to the severity of hematopoietic failure. From the perspective of a comprehensive understanding of the disease, domestic staging has its unique advantages.
It is important to point out that the peripheral blood triad of peripheral blood cells shows a parallel decline in typical reblasts, but in some special cases, such as early reblasts, this feature may not be obvious and often manifests first as thrombocytopenia and neutropenia. In patients with anemia, if the platelet count is still normal, it is important to note that it may be due to other disorders rather than reoccurrence; the absolute peripheral blood reticulocyte count in patients with reoccurrence should be emphasized.
In patients with anemia, peripheral blood reticulocyte counts of at least 100×109/L can be considered as effective bone marrow compensation, while in patients with reoccurrence, bone marrow hematopoiesis is not compensated and reticulocyte counts are relatively or absolutely reduced; it is often unreliable to diagnose or exclude reoccurrence with the results of a single bone marrow aspiration smear from a particular site, and the results of multiple bone marrow aspirations from different sites can more objectively reflect systemic bone marrow hematopoiesis; modern diagnosis of reoccurrence must Include bone marrow biopsy.
In order to evaluate the area of bone marrow hematopoiesis and to make the necessary differential diagnosis, the evaluation of hypoplasia and steatosis of bone marrow must take into account the influence of age factors, and the evaluation of hypoplasia of bone marrow in the elderly must be carried out simultaneously with peripheral blood examination or multiple bone marrow aspiration examinations at multiple sites. It is important to predict disease progression from a dynamic and developmental viewpoint and make a reasonable typing diagnosis at an early stage, especially for acute reoccurrence.
Second, the clinical diagnosis of reoccurrence: clinically, the possibility of AA should be considered in patients with whole blood cytopenia, and the diagnosis is generally not difficult in typical cases, but in atypical cases, such as early cases, where clinical manifestations and laboratory tests are not yet obvious, or AA combined or superimposed with other clinical conditions, the diagnosis can also be difficult.
As with other diseases, the diagnosis of AA requires a detailed history, a thorough physical examination, and the necessary ancillary tests. The medical history should emphasize the history of occupation, exposure to chemical and radioactive substances, and the drugs applied within 6 months before the onset of the disease should be recorded in detail. Any clinical manifestation of progressive anemia, bleeding and susceptibility to infection, such as complete blood cytopenia, and no enlargement of liver, spleen and lymph nodes on examination, should be considered as possible AA. Congenital aplastic anemia, including Fanconi anemia and congenital dyskeratosis, must be suspected in children and young patients with developmental delay, malformation, skin pigmentation, mucous membrane leukoplakia, and nail dystrophy.
The significance of hematological examination for the diagnosis of this disease is beyond doubt. A complete cell count, including reticulocyte count, should be performed in peripheral blood. Bone marrow examination should include bone marrow fluid smear and bone marrow biopsy, which are the most important basis for the diagnosis of the disease. In cases of clinically suspected aplastic anemia with atypical bone marrow examination, multiple punctures and biopsies from multiple sites should be performed.
In addition, liver function, virology, serum folate and vitamin B12 and autoantibody tests are also required. Flow cytometry for paroxysmal sleep hemoglobinuria (PNH) microclones and cytogenetic testing of peripheral blood and bone marrow can help further confirm the diagnosis of aplastic anemia and exclude other disorders with similar clinical and laboratory manifestations. Diepoxybutane-induced chromosome fragility test (DEB test) should be routinely performed in children and young patients under 50 years of age to exclude Fanconi anemia.
The British Committee for Standards in Haematology recommends the following tests for the diagnosis of reanemia:
1. Complete blood count and reticulocyte count
2. peripheral blood smear
3. HbF% test for children
4.Bone marrow aspiration smear, bone marrow biopsy and chromosome examination of bone marrow hematopoietic cells
5.Patients younger than 50 years old should have peripheral blood chromosome breakage analysis to exclude Fanconi anemia
6.Flow cytometry for GPI anchor protein
7. Urine ferritin test for abnormal GPI anchor protein or Ham test
8.Serum folate and vitamin B12 concentration measurement
9.Liver function
10.Virus testing, including hepatitis A virus, hepatitis B virus, hepatitis C virus, EBV, HIV, CMV, etc.
11.Anti-nuclear antibody and anti-dsDNA
12.Chest x-ray examination
13.Abdominal ultrasound and echocardiography
14.If the patient is clinically compatible but does not respond to immunosuppressive therapy, peripheral blood telomere length testing or telomere related gene mutation analysis should be performed to exclude congenital dyskeratosis.
AA may occur in certain specific physiological or pathological conditions, or may be complicated by other disorders, and its presentation may not be typical. For example, AA during pregnancy, AA associated with hepatitis, AA complicated by Silhan syndrome, AA complicated by tuberculosis, etc. In addition to anemia, hemorrhage and infection, AA also shows symptoms, signs and laboratory features related to the complications, which are not difficult to recognize by careful examination.
However, because the kidneys secrete more erythropoietin during anemia, resulting in the release of young bone marrow red blood cells to peripheral blood earlier, in fact, the average volume of peripheral blood red blood cells in most patients with reoccurrence is at the upper limit of normal value or mildly increased. In fact, most patients with reoccurring disorders have a mean peripheral blood erythrocyte volume at the upper limit of normal or mildly increased. The presence of nucleated erythrocytes in peripheral blood smears in patients with untreated reoccurrence is usually rare, and the presence of nucleated erythrocytes in peripheral blood requires careful questioning of prior treatment history, suspicion of other diseases and careful differentiation.
The absolute value of reticulocytes is emphasized. A normal or even mildly elevated ratio of reticulocytes in uncorrected peripheral blood should not be used as a basis to exclude the diagnosis of reoccurrence, and absolute reticulocyte counts below 100×109/L in anemic patients should be considered as possible bone marrow hematopoietic failure. Therefore, a single bone marrow aspiration smear is often not a true and objective reflection of bone marrow hematopoiesis, especially based on the active/apparently active proliferation of nucleated cells in the sternal bone marrow aspiration smear, or even the non-reduction of megakaryocyte count to exclude the diagnosis of reoccurrence; the more important purpose of sternal bone marrow aspiration smear is to perform The more important purpose of sternal bone marrow aspiration is to make a differential diagnosis and to evaluate the severity of hematopoietic failure.
Young erythrocytes of the bone marrow may show mild morphological changes of abnormal development, while granulocyte and megakaryocyte morphology are mostly not significantly abnormal. To evaluate myelopoietic hematopoiesis for the differential diagnosis of reblastoma and myelodysplastic syndrome (MDS), it is important to consider the effect of treatment response and therapeutic agents, especially cell growth factors (e.g., EPO, G-CSF, etc.), on the morphologic changes of cells.
The diagnostic value of bone marrow granules and bone marrow fat droplets for the diagnosis of remitting disease should be emphasized. Significant bone marrow steatosis incompatible with age as well as empty bone marrow granules and a marked increase in non-hematopoietic cells tend to favor the diagnosis of remitting disease. The bone marrow fluid is thin with increased oil droplets, but rarely difficult to aspirate by puncture, and bone marrow “dry aspiration” should be noted for bone marrow fibrosis or bone marrow metastases, etc. Bone marrow biopsy requires good bone marrow tissue specimens at least 1 to 2 cm long. In addition to routine staining of bone marrow tissue structure, degree of nucleated cell proliferation, assessment of the proportion of each lineage and abnormal cell examination, immunohistochemical staining with antibodies such as CD34, CD117, CD68, myeloperoxidase and lysozyme can help to differentiate rechondroplasia from MDS.
An increased percentage of CD34+ cells in the bone marrow and the tendency of CD34+ cells to form clusters/clusters suggest that hematopoietic failure is due to MDS rather than remitting disease. 50% of patients with remitting disease and some patients with intermediate and low-risk MDS may have microscopic PNH clones without clinical manifestations of hemolysis, and flow cytometry detection of the absence of the hematopoietic anchor proteins CD55 and CD59 can help evaluate the size of PNH clones. The presence of micro PNH clones and the detection of PIG-A mutations based on sensitive molecular biology methods cannot distinguish between reoccurring disorders and MDS. reoccurring disorders have a significant reduction in bone marrow hematopoietic cells, making it difficult to obtain sufficient cytodifferentiation images, which can easily lead to failure of routine cytogenetic tests.
Differentiation between reoccurrence and congenital bone marrow hematopoietic failure: Many other hematologic and non-hematologic diseases may have similar clinical manifestations to reoccurrence and need to be differentiated, but it is not difficult to distinguish them after careful history taking, physical examination and corresponding laboratory tests. In children and young adults, the prognosis is not only different between acquired remittent disease and congenital hematopoietic failure, but also the treatment strategies and therapies are significantly different, and the differentiation between the two is sometimes exceptionally difficult.
Congenital myelopoietic failure is a rare group of genetically heterogeneous disorders characterized by congenital somatic anomalies, myelopoietic failure and susceptibility to neoplasia, manifested by peripheral blood allogeneic cytopenia and reduced myelopoietic cell proliferation, mainly in Fanconi anemia (FA) and congenital dyskeratosis (DC). . Patients with somatic abnormalities are less likely to be missed, but approximately 20% of FA patients may have no somatic abnormalities, and some patients may not start to develop hematologic changes in their reperfusion until adulthood (the maximum age of onset has been reported to be 49 years), making them vulnerable to underdiagnosis and misdiagnosis. The upper age limit for screening has been revised upwards from 35 years to 50 years.
Cells from FA patients show spontaneous chromosome breaks and are highly sensitive to DNA cross-linking agents such as diepoxybutane (DEB) and mitomycin (MMC), and chromosome breaks in peripheral blood lymphocytes from FA patients treated with DEB and MMC are significantly increased, which is the current gold standard for the diagnosis of FA. Chromosome breakage test can be used for peripheral blood or bone marrow cells, amniocytes, chorionic villous cells, fetal blood cells and skin fibroblasts.
In addition, lymphocytes in FA patients are blocked in the G2/M phase and the G2 phase is significantly prolonged. The application of flow cytometry to detect the cell cycle suggests their accumulation in the G2/M phase, which can be used for the diagnosis of FA. Application of immunoblotting assay, any protein deletion in the FANCD2-L core complex will not detect FANCD2-L, enabling rapid examination of protein deletion in the core complex for diagnosis of FA and preliminary determination of possible FA types. The comet test is a single-cell gel electrophoresis for rapid detection of DNA damage and can also be used for FA patients and carriers.
In patients with normal lymphocyte DEB test or MMC test and high clinical suspicion of FA, further fibroblast DEB test or MMC test should be performed to exclude false negative lymphocyte DEB test due to FA cell mutations that cause somatic cells to recover (sic recovery) to normal.
Patients with typical DC often show a triad of signs: toenail dyskeratosis, skin hyperpigmentation, and oral mucosal leukoplakia, which is easier to diagnose; patients who show bone marrow failure without obvious physical abnormalities are more difficult to diagnose. Telomere length of peripheral blood subsets of leukocytes by flow cytometry and fluorescence in situ hybridization is currently the best diagnostic method [5], and the combined analysis of telomere length of leukocyte subsets (total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells) is highly sensitive and specific for the diagnosis of DC, and can be used to differentiate it from other bone marrow hematopoietic failures.
There are four genes associated with the development of DC: DKC1 gene, which is X-linked, TERC and TERT, which are autosomal dominant, and NOP10, which is autosomal recessive, and the detection of mutations in these genes will be more helpful for the diagnosis of DC. In view of the fact that most units do not perform leukocyte telomere length test and DC-related gene mutation test as a routine clinical examination, it is recommended to perform this test at least in patients with ineffective immunosuppressive therapy to help clarify the diagnosis and develop a re-treatment plan.
V. Reminiscence with hypoproliferative MDS and hypoproliferative leukemia: The literature reports that 63%, 24%, 13% and 36% of patients with AML and MDS have markedly active and extremely active (Hypercellular), active (Normocellular) and hypoproliferative (Hypocellular) bone marrow proliferation, respectively. Even after age correction, AML and MDS with hypoproliferative bone marrow still accounted for 2.2% and 7%, respectively.
Hypoproliferative AML and hypoproliferative MDS also often show marked peripheral cytopenia, and conventional morphologic criteria based on morphologic identification of developmental abnormalities and primitive cell % counts are very difficult to use to differentiate them from reperfusion because of the marked reduction of nucleated cells in the bone marrow. The FAB Collaborative Group recommends the following methods for differentiating hypoproliferative AML/MDS from remittent disease (Table 2).
1. A peripheral blood smear with clear primitive granulocytes should be considered hypoproliferative MDS or AML.
2, The proportion of granulocytic pathological hematopoiesis (e.g., granulocytopenia, pseudo-Pelger aberration) in the bone marrow, which exceeds 10% of granulocytes, should be considered hypoproliferative MDS or AML.
3. If pathological hematopoiesis is present in the bone marrow and the primitive granulocytes are between 1% and 20%, the diagnosis is MDS;
4. The granulocytic and megakaryocytic lineages in AA bone marrow cannot show morphological abnormalities, but the red lineage allows moderate pathological hematopoiesis, such as multinucleated erythrocytes, H-J vesicles visible in the cytoplasm, and intercellular bridges (intercellular bridges are a manifestation of cytopathic hematopoiesis – this is an explanation and will not be included in the text), etc.
5. The presence of ring-shaped iron-granulated juvenile erythrocytes in the bone marrow (>5 granules in the perinuclear area or >1/3 around the perinuclear area) is a manifestation of pathological hematopoiesis in the red lineage of the bone marrow and cannot be diagnosed as AA.
6. A validated bone marrow biopsy of at least 1 to 2 CM of bone marrow tissue must be obtained to ensure the differentiation of reperfusion or other bone marrow failure disorders under a 100x light microscope.
7. Bone marrow biopsies detecting more than 2 primitive cell clusters (at least 3 or more primitive cells) growth should be considered as MDS or AML.
For patients with peripheral whole blood cytopenia with bone marrow hematopoietic failure, careful history taking, physical examination, and necessary laboratory tests to exclude other diseases causing similar manifestations are necessary to diagnose reoccurrence. After the diagnosis of reoccurrence is clear, further diagnosis of reoccurrence severity should be made based on peripheral blood parameters and bone marrow hematopoietic area in order to establish a treatment plan. Patients with abnormal cytogenetic clones or microscopic PNH clones should be monitored for a long period of time to alert for disease transformation. Both children and young adults with reocclusion should be subtyped with congenital bone marrow hematopoietic failure as much as possible, and hematopoietic stem cell transplantation is recommended for heavy and very heavy patients. For patients with reoccurrence that cannot be fully diagnosed or whose cases are atypical, a consultation with a multidisciplinary expert may be requested.
Table 1 Severity of reoccurrence staging
Diagnostic criteria
SAA
Bone marrow
Cell area < 25% of normal; if < 50% of normal, hematopoietic cells should be < 30%
Peripheral blood indicators
At least two of the following: granulocytes < 0.5 X 109 /L; corrected reticulocytes < 1%, or reticulocytes < 20 X 109 /L in absolute value; platelets < 20 X 109 /L.
Very heavy reperfusion (VSAA)
Neutrophils < 0.2 X 109 /L, remaining criteria as for SAA
Non-severe reoccurrence (NSAA)
SAA criteria were not met
Table 2. Differential diagnosis of reoccurrence
Peripheral blood smear
Counting classification 100 cells
Count the percentage of granulocytes with abnormal development
Check for the presence of primitive granulocytes
Bone marrow examination
Counting of 500 cells
Check for granulocytes, erythrocytes, and megakaryocytes and count the proportion of these cells in the lineage
Iron staining to check for the presence of ringed iron granulocytes
Bone marrow biopsy
To examine the cellular composition of the bone marrow
Check for primitive cell displacement (ALIP), immunohistochemistry recommended: CD34, CD117, MPO
Reticulin staining for the presence of myelofibrosis
Other tests
FISH for cytogenetics
Flow cytometry examination
Examination of PNH clone