Guidelines for the diagnosis and management of acquired aplastic anaemia

The purpose of this guideline is to provide a rational approach to the investigation and management of patients with acquired aplastic anaemia. These guidelines have been produced by both specialists in the field of aplastic anaemia and experienced district general hospital haematologists, and reviewed by members of the British Committee for Standards in Haematology (BCSH) General Haematology Task Force. Because aplastic anaemia is a rare disease, many of the statements and comments in the first part of this manuscript are based on review of the literature and expert or consensus opinion rather than on clinical studies or trials. Medline, Cinahl and Embase databases were searched for this purpose. Levels of evidence for treatment of aplastic anaemia also reflect the rarity of this condition. To ensure wide dissemination of these guidelines, they are also available on the BCSH website and will be reviewed on a three yearly basis. Aplastic anaemia is defined as pancytopenia with a hypocellular bone marrow in the absence of an abnormal infiltrate and with no increase in reticulin. For these guidelines we will focus specifically on idiosyncratic acquired aplastic anaemia, and will not refer to the inevitable and predictable aplasia that occurs after chemotherapy and/or radiotherapy. The incidence of acquired aplastic anaemia in Europe and North America is around 2 per million population per year. The incidence is two to three times higher in East Asia. There is a biphasic age distribution with peaks from 10–25 to >60 years. There is no significant difference in incidence between males and females (Heimpel, 2000). Congenital aplastic anaemia is very rare, the commonest type being Fanconi's anaemia which is inherited as an autosomal recessive disorder. Patients with aplastic anaemia most commonly present with symptoms of anaemia and skin or mucosal haemorrhage or visual disturbance due to retinal haemorrhage. Infection is a less common presentation. There is no lymphadenopathy or hepatosplenomegaly (in the absence of infection) and these findings strongly suggest another diagnosis (Gordon-Smith, 1991). In children and young adults, the findings of short stature, café au lait spots, and skeletal anomalies should alert the clinician to the possibility of a congenital form of aplastic anaemia, Fanconi's anaemia, although Fanconi's anaemia can sometimes present in the absence of overt clinical signs. Patients with Fanconi's anaemia most commonly present between the ages of 3 and 14 years but can occasionally present later in their 30s (up to 32 in males and 48 years in females reported by Young & Alter, 1994). The findings of leucoplakia, nail dystrophy and pigmentation of the skin are characteristic of another inherited form of aplastic anaemia, dyskeratosis congenita, with a median age at presentation of 7 years (range 6 months to 26 years) (Dokal, 2000). A preceding history of jaundice, usually 2–3 months before, may indicate a posthepatitic aplastic anaemia (Gordon-Smith, 1991; Young & Alter, 1994). Many drugs and chemicals have been implicated in the aetiology of aplastic anaemia, but for only very few is there reasonable evidence for an association from case–control studies, and even then it is usually impossible to prove causality (Baumelou et al, 1993; Young & Alter, 1994; Heimpel, 1996; Kauffmann et al, 1996; Issaragrissil et al, 1997) (see Table I). A careful drug history should be obtained detailing all drug exposures for a period beginning 6 months and ending 1 month prior to presentation (Heimpel, 1996; Kauffmann et al, 1996). If, at presentation, the patient is taking several drugs that may have been implicated in aplastic anaemia, even if the evidence is based on case report(s) alone, then all the putative drugs should be discontinued and the patient should not be re-challenged with the drugs at a later stage after recovery of the blood counts. The Committee on Safety of Medicines (CSM) should be informed using the Yellow Card Scheme on every occasion that a patient presents with aplastic anaemia where there is a possible drug association. Similarly, a careful occupational history of the patient may reveal exposure to chemicals or pesticides that have been associated with aplastic anaemia, as summarized in Table II. The following investigations are required to (i) confirm the diagnosis, (ii) exclude other possible causes of pancytopenia with a hypocellular bone marrow, (iii) exclude congenital aplastic anaemia, (iv) screen for an underlying cause of acquired aplastic anaemia, and (v) document or exclude a co-existing abnormal cytogenetic clone or a paroxysmal nocturnal haemoglobinuria (PNH) clone. Investigations for the diagnosis of aplastic anaemia are presented in Table III. The full blood count (FBC) typically shows pancytopenia although usually the lymphocyte count is preserved. In most cases the haemoglobin level, neutrophil and platelet counts are all uniformly depressed, but in the early stages isolated cytopenia, particularly thrombocytopenia, may occur. Anaemia is accompanied by reticulocytopenia, and macrocytosis is commonly noted. Careful examination of the blood film is essential to exclude the presence of dysplastic neutrophils and abnormal platelets, blasts and other abnormal cells such as hairy cells. The monocyte count may be depressed but the absence of monocytes should alert the clinician to a possible diagnosis of hairy cell leukaemia. In aplastic anaemia, anisopoikilocytosis is common and neutrophils may show toxic granulation. Platelets are reduced in number and mostly of small size. Fetal haemoglobin (HbF) should be measured pretransfusion in children, as this is an important prognostic factor in paediatric myelodysplastic syndrome (MDS) that may feature in the differential diagnosis of pancytopenia in children. Both a bone marrow aspirate and trephine biopsy are required. Fragments are usually readily obtained from the aspirate. Difficulty obtaining fragments should raise the suspicion of a diagnosis other than aplastic anaemia. The fragments and trails are hypocellular with prominent fat spaces and variable amounts of residual haemopoietic cells. Erythropoiesis is reduced or absent, dyserythropoiesis is very common and often marked, so this alone should not be used to make a diagnosis of MDS. Megakaryocytes and granulocytic cells are reduced or absent; dysplastic megakaryocytes and granulocytic cells are not seen in aplastic anaemia. Lymphocytes, macrophages, plasma cells and mast cells appear prominent. In the early stages of the disease, one may also see prominent haemophagocytosis by macrophages, as well as background eosinophilic staining representing interstitial oedema. A trephine is crucial to assess overall cellularity, to assess the morphology of residual haemopoietic cells and to exclude an abnormal infiltrate. In most cases the trephine is hypocellular throughout but sometimes it is patchy, with hypocellular and cellular areas. Thus, a good quality trephine of at least 2 cm is essential. A ‘hot spot’ in a patchy area may explain why sometimes the aspirate is normocellular. Care should be taken to avoid tangential biopsies as subcortical marrow is normally ‘hypocellular’. Focal hyperplasia of erythroid or granulocytic cells at a similar stage of maturation may be observed. Sometimes lymphoid aggregates occur, particularly in the acute phase of the disease or when the aplastic anaemia is associated with systemic autoimmune disease such as rheumatoid arthritis or systemic lupus erythematosus. The reticulin is not increased and no abnormal cells are seen. Blasts are not seen in aplastic anaemia, and their presence either indicates a hypocellular MDS or evolution to leukaemia (Tichelli et al, 1992; Marin, 2000). To define aplastic anaemia there must be at least two of the following: (i) haemoglobin <10 g/dl, (ii) platelet count <50 × 109/l, (iii) neutrophil count <1·5 × 109/l (International Agranulocytosis and Aplastic Anaemia Study, 1987). The severity of the disease is graded according to the blood count parameters and bone marrow findings as summarized in Table IV (Camitta et al, 1976; Bacigalupo et al, 1988). The assessment of disease severity is important in treatment decisions and has prognostic significance. Patients with bi- or trilineage cytopenias that are less severe than this are not classified as aplastic anaemia. However, they should have their blood counts monitored to determine whether they will develop aplastic anaemia with time. Liver function tests should be performed routinely to detect antecedent hepatitis. In posthepatitic aplastic anaemia the serology is most often negative for all the known hepatitis viruses. The onset of aplastic anaemia occurs 2–3 months after an acute episode of hepatitis and is more common in young males (Brown et al, 1997). Blood should be sent for hepatitis A antibody, hepatitis B surface antigen, hepatitis C antibody and Epstein–Barr virus (EBV) screens. Cytomegalovirus (CMV) and other viral serology should be assessed if bone marrow transplantation (BMT) is being considered. Parvovirus causes red cell aplasia but not aplastic anaemia. Vitamin B12 and folate levels should be measured to exclude megaloblastic anaemia which, when severe, can present with pancytopenia. If a deficiency of vitamin B12 or folate is documented, this should be corrected before a final diagnosis of aplastic anaemia is confirmed. The occurrence of pancytopenia in systemic lupus erythematosus may (i) be autoimmune in nature occurring with a cellular bone marrow or (ii) be associated with myelofibrosis or rarely, (iii) occur with a hypocellular bone marrow. Blood should be sent for anti-nuclear antibody and anti-DNA antibody in all patients presenting with aplastic anaemia. Paroxysmal nocturnal haemoglobinuria should be excluded by performing a Ham test and/or flow cytometry (Lewis et al, 2001). Analysis of phosphatidylinositol glycan (PIG)-anchored proteins, such as CD55 and CD59, by flow cytometry, however, is the much more sensitive test for PNH, enabling the detection of small PNH clones which occur in at least 20–25% of patients with aplastic anaemia (Dunn et al, 1999; Socie et al, 2000). Such small clones are most easily identified in the neutrophil and monocyte lineages in aplastic anaemia and will be detected by flow cytometry but not by the Ham test. If the patient has had a recent blood transfusion, the Ham test may be negative whereas a population of PIG deficient red cells may still be detected by flow cytometry. However, the clinical significance of a small PNH clone in aplastic anaemia as detected by flow cytometry remains uncertain. Such clones can remain stable, diminish in size, disappear or increase. What is clinically important is the presence of a significant PNH clone with clinical or laboratory evidence of haemolysis, and this will be detected by the Ham test. Urine should be examined for haemosiderin to exclude intravascular haemolysis which is a constant feature of haemolytic PNH. Evidence of haemolysis associated with PNH should be quantified with the reticulocyte count, serum bilirubin, serum transaminases and lactate dehydrogenase (LDH). Peripheral blood lymphocytes should be examined cytogenetically in all patients under the age of 35 years and probably also up to the age of 45 for older patients who are potential BMT candidates, for spontaneous and diepoxybutane (or mitomycin-C)-induced increase in chromosome breakages and aberrations characteristic of Fanconi's anaemia (Young & Alter, 1994). Cytogenetic analysis of the bone marrow should be attempted although this may be difficult in a very hypocellular bone marrow and often insufficient metaphases are obtained. In this situation, one should consider fluorescent in situ hybridization analysis for chromosomes 5 and 7 in particular. It was previously assumed that the presence of an abnormal cytogenetic clone indicated a diagnosis of MDS and not aplastic anaemia, but it is now evident that abnormal cytogenetic clones may be present in up to 11% of patients with otherwise typical aplastic anaemia at diagnosis (Appelbaum et al, 1989; Tichelli et al, 1996). The presence of abnormal cytogenetics at presentation in children, especially monosomy 7, should alert to the likelihood of MDS. Abnormal cytogenetic clones may also arise during the course of the disease (Socie et al, 2000). The management of a patient with aplastic anaemia who has an abnormal cytogenetic clone is discussed in ‘Management of aplastic anaemia in the presence of an abnormal cytogenetic clone’. A chest X-ray (CXR) is useful at presentation to exclude infection and for comparison with subsequent films. Routine X-rays of the radii are no longer indicated as all young patients should have peripheral blood chromosomes sent to exclude a diagnosis of Fanconi's anaemia. Abdominal ultrasound: the findings of an enlarged spleen and/or enlarged lymph nodes raise the possibility of a malignant haematological disorder as the cause of the pancytopenia. In younger patients, abnormal or anatomically displaced kidneys are features of Fanconi's anaemia. The above investigations should exclude causes of a hypocellular bone marrow with pancytopenia other than aplastic anaemia. These include: Hypocellular MDS/acute myeloid leukaemia (AML) can sometimes be difficult to distinguish from aplastic anaemia. The following features of MDS are not found in aplastic anaemia: dysplastic cells of the granulocytic and megakaryocytic lineages, blasts in the blood or marrow (Tuzuner et al, 1995; World Health Organisation Classification of Tumours, 2001). In trephine specimens, increases in reticulin associated with residual areas of haemopoiesis suggest hypocellular MDS rather than aplastic anaemia. The presence of abnormal localization of immature precursors (ALIPs) is difficult to interpret in this context because small collections of immature granulocytic cells may be seen in the bone marrow in aplastic anaemia when regeneration occurs. As discussed previously, dyserythropoiesis is very common in aplastic anaemia. Hypocellular acute lymphoblastic leukaemia (ALL) occurs in 1–2% of cases of childhood ALL. Overt ALL usually develops within 3–9 months of the apparent bone marrow failure. In contrast to aplastic anaemia, the neutropenia is usually more pronounced than the thrombocytopenia and sometimes there is an increase in reticulin within the hypocellular bone marrow (Chessells, 2001). For all new paediatric cases of aplastic anaemia, a national central morphology review is planned under the aegis of the Medical Research Council Childhood Leukaemia Working Party Subgroup for rare haematological diseases. Hairy cell leukaemia classically presents with pancytopenia but the accompanying monocytopenia is a constant feature of this disorder. It is usually difficult or impossible to aspirate on bone marrow fragments. In addition to the typical interstitial infiltrate of hairy cells with their characteristic ‘fried egg’ appearance in the bone marrow trephine, there is always increased reticulin. Immunophenotyping reveals CD20+, CD11c+, CD25+, FMC7+, CD103+ tumour cells which are typically CD5−, CD10− and CD23−. Although splenomegaly is a common finding in hairy cell leukaemia, it may be absent in 30–40% of cases (Catovsky, 1999). Lymphomas, either Hodgkin's disease or non-Hodgkin's lymphoma, and myelofibrosis may sometimes present with pancytopenia and a hypocellular bone marrow. The bone marrow biopsy should be examined very carefully for foci of lymphoma cells or fibrosis which may be seen in only a small part of the trephine. Myelofibrosis is usually accompanied by splenomegaly and the absence of an enlarged spleen in the presence of marrow fibrosis should alert one to secondary malignancy. Marker studies and gene rearrangement studies will help confirm the diagnosis of lymphoma. Mycobacterial infections can sometimes present with pancytopenia and a hypocellular bone marrow, this is seen more commonly with atypical mycobacteria. Other bone marrow abnormalities include granulomas, fibrosis, marrow necrosis and haemophagocytosis. Demonstrable acid alcohol fast bacilli (AAFB) and granulomas are often absent in Mycobacterium tuberculosis infection. AAFB are more frequently demonstrated in atypical mycobacterial infections where they are often phagocytosed by foamy macrophages. The bone marrow aspirate should be sent for AAFB culture if tuberculosis is suspected (Bain et al, 2001). Anorexia nervosa or prolonged starvation may be associated with pancytopenia. The bone marrow may show hypocellularity and gelatinous transformation (serous degeneration/atrophy) with loss of fat cells as well as haemopoietic cells, and increased ground substance which stains a pale pink on haematoxylin/eosin stain (Bain et al, 2001). The pink ground substance may also be seen as on a May–Grünwald–Giemsa stained aspirate. Some degree of fat change may also be seen in aplastic anaemia, especially early in its evolution. Consideration should be given to review blood and bone marrow slides by a specialist centre, especially if there are unusual morphological features or where there is any doubt about the diagnosis (see also ‘Specific treatment of aplastic anaemia’ relating to further specialist advice on the specific treatment of aplastic anaemia). Support with red cell and platelet transfusions is essential for patients with aplastic anaemia to maintain a safe blood count. It is recommended to give prophylactic platelet transfusions when the platelet count is <10 × 109/l (or <20 × 109/l in the presence of fever) (Grade C recommendation; level IV evidence, Appendix 1), rather than giving platelets only in response to bleeding manifestations (Murphy et al, 1992; Norfolk et al, 1998). Prediction of bleeding is difficult in an individual patient. Fatal haemorrhage, usually cerebral, is more common in patients who have <10 × 109/l platelets, extensive retinal haemorrhages, buccal haemorrhages or rapidly spreading purpura. However, cerebral haemorrhage may be the first major bleed in patients who have none of these other bleeding manifestations (Gordon-Smith, 1991). A common problem in multi-transfused patients with aplastic anaemia, compared with leukaemia patients, is that they may develop alloimmunization to leucocytes present in red cell and platelet transfusions by generating human leucocyte antigen (HLA) or non-HLA (minor histocompatibility) antibodies. This can result in platelet refractoriness, as well as an increased risk of graft rejection after allogeneic BMT (Kaminsky et al, 1990). Other causes of platelet refractoriness should also be excluded, namely sepsis and drugs such as amphotericin and vancomycin). Routine prestorage leucocyte depletion of all units of red cells and platelets in the UK is likely to reduce the risk of alloimmunization (Killick et al, 1997; Ljungman, 2000). In a retrospective, single centre study, the incidence of HLA alloimmunization was reported to be 50% in patients with aplastic anaemia who had received blood products prior to the introduction of prestorage leucocyte depletion in the UK compared with only 12% for patients who received only leucocyte-depleted blood products (Killick et al, 1997). Patients who become refractory to platelet transfusions should be screened for HLA antibodies. However, other causes of platelet refractoriness such as infection and drugs should be excluded. If a patient does become sensitized to random donor platelets resulting in platelet refractoriness, HLA-matched platelets should be used instead. Red cell and platelet transfusions should be given to maintain a safe haemoglobin level and platelet count and not be withheld for fear of sensitizing the patient. Directed blood and platelet donations from family members are not permitted within the National Blood and the may become sensitized to from the potential bone marrow donor resulting in a risk of graft In a family donor may provide the most platelets if a patient has HLA and platelets from platelet other important to help bleeding include good the of acid and of with If a patient is a potential for early or later BMT (see donor it is recommended that the patient is with only negative blood products the is negative blood products should then be only if both the patient and donor are negative et al, 1999). There are to suggest that of all red cell and platelet transfusions before BMT further the risk of to reduced risk of graft rejection after allogeneic et al, 1994). Although an expert on aplastic anaemia that blood products should be used routinely in all patients with aplastic anaemia who are et al, such is not recommended in the UK Blood Task where the leucocyte depletion of all blood and platelet transfusions may be at least as important in the risk of The for red cell and platelet transfusions from the beginning of the to all patients cell transfusions were frequently used in the early to help infection in patients, but are now not indicated in of their of and risk of 2000). However, transfusions may make a in the with the possibility of of from factor et al, 2000). There are no and safe haemopoietic to red cells and platelets in patients with aplastic anaemia (see for a general of in aplastic anaemia has that it is which is not in of the of serum levels in the of patients with aplastic anaemia. A of using is the potential for severe and or of anaemia due to red cell aplasia from et al, in with other drugs used routinely to aplastic anaemia, such as there is the potential for The of in aplastic anaemia is not recommended (Grade C recommendation; level IV Other haemopoietic have been used in aplastic anaemia to determine whether they was in a study, but the was early because of severe anaemia and the onset of haemorrhage in patients with aplastic anaemia et al, In a small study, cell factor was to trilineage haemopoiesis in patients with aplastic anaemia et al, but its in a with and was because of from 2001). There have been no clinical studies of human in aplastic anaemia. The of the of and factor in prolonged thrombocytopenia and of its in clinical 2000). The of and factor in aplastic anaemia is discussed in further later (see of The risk of infection is by the neutrophil and monocyte counts et al, et al, The risk may also be on an individual as patients have infections may have none or very Patients with aplastic anaemia are at risk of and infections 2000). infections have a very in patients with severe aplastic anaemia because of the prolonged of severe neutropenia Patients with a risk of infection should be in when in hospital and should prophylactic and an such as and of (Gordon-Smith, 1991; Ljungman, 2000). are not essential but should be used when are given to help either a of two such as and or a such as However, there is about the of and an increase in In be used to if it is used The of either or should be to individual For children, it is not to prophylactic is not and are very drugs such as and amphotericin have been by prophylactic However, one may consider using especially if there is a history of infection as is The of is and and more serum levels et al, 1999). Liver can also be a problem with in clinical may prove to be more as prophylactic There is no for prophylactic or in patients with aplastic anaemia. with is essential for all patients and with is essential but is not indicated following treatment 2000). For patients who are in the and who have not received or with an is but prophylactic are not required in all For patients who are very count × prophylactic and should be used and that may be with or It is less whether and should for at risk of infection count × The is on an individual according to the and severity of As for all patients, may and treatment before the of investigations are The hospital guidelines for treatment of neutropenia should be This most frequently a of such as an and a the on hospital The of the infection history and recent will also the the early introduction of It is recommended that amphotericin is the neutropenia early if a patient with aplastic anaemia is with it is usually difficult to as the neutrophil count may not for a period of time. If a patient has had or if infection is or even amphotericin should be used with the first (or of an of amphotericin or one of the such as or should be in aplastic anaemia patients who may prolonged in to avoid and infection should be taken as of likely infection in patients with severe aplastic anaemia A should be as part of the investigation of new or with of the chest if there is about the Although there have been no studies the of or other haemopoietic in the treatment of severe infection in patients with aplastic anaemia. A short course of at a of 5 may be for severe systemic infections that are not to and It may a neutrophil