The peripheral blood film shows pancytopenia without gross morphological abnormalities in the circulating cells. There may be some macrocytosis of remaining red cells, usually with an absolute reticulocytopenia. A relative reticulocytosis should always raise the possibility of associated paroxysmal nocturnal haemoglobinuria (PNH). Granulocytes often show increased staining of granules, the so-called toxic granulation of neutropenia. Monocytes are usually reduced in proportion to the granulocytes. Platelets are reduced and of small and uniform size. There is a variable reduction in the lymphocyte count between individuals; the count is sometimes normal or even increased so that the total white cell count is normal or near normal but, more commonly, the lymphocyte count is reduced. Abnormal cells are not seen. The bone marrow aspirate is normally easily obtained, typically with many fragments, which appear hypo-cellular (Figure 13.1a). The cell trails are hypocellular, with a relative increase in lymphocytes and plasma cells and other non-haemopoietic forms. Remaining haemopoietic precursors are normal in appearance.

In the early stages of aplastic anaemia, haemophagocytosis may be prominent. In a high proportion of cases, the hypocellu-larity of the marrow is patchy, with quite extensive areas of cellular marrow remaining. The bone marrow aspirate may, under these circumstances, be misleadingly cellular. A trephine biopsy (sometimes more than one) is necessary to assess cellularity properly.

The bone marrow trephine shows the fat replacement of marrow with or without the remaining islands of cellularity (Figure 13.1b and c). Non-haemopoietic cells remain, sometimes giving the impression of a chronic inflammatory infiltrate. Reticulin is not increased. The most common mistake in the diagnosis of aplastic anaemia is to make the diagnosis on the basis of a bone marrow aspirate in the presence of pancytopenia without obtaining adequate trephine specimen. Other conditions that can also present with pancytopenia and a hypocellular bone marrow include hypocellular myelodysplastic syndrome, hypo-cellular acute myeloid leukaemia, hypocellular acute lympho-blastic leukaemia, hairy cell leukaemia, lymphoma, myelofibrosis, mycobacterial infections and anorexia nervosa or prolonged starvation, emphasizing the importance of careful examination of not only the bone marrow but also a well-stained peripheral blood film.

Cytogenetic analysis of the bone marrow should be performed, although it is often difficult to obtain sufficient metaphases in a very hypocellular bone marrow. Abnormal cytogenetic clones occur in up to 11% of patients with otherwise typical aplastic anaemia, and their presence does not necessarily indicate a diagnosis of myelodysplastic syndrome. The most frequently observed abnormalities include trisomy 8, trisomy 6, 5q- and anomalies of chromosomes 7 and 13. Often the abnormal clone is small, constituting only a small proportion of total metaphases, and not infrequently it may be transient and disappear spontaneously or after haematological response to immunosuppressive therapy.

Peripheral blood lymphocytes should also be examined cyto-genetically in all patients under the age of 35 years for spontaneous and di-epoxybutane (or mitomycin C)-induced increase

Figure 13.1 Aplastic anaemia. (a) Bone marrow aspirate showing fragments with typical lacy appearance and fatty hypocellular trails (x80). (b) Bone marrow trephine biopsy showing absence of haemopoietic tissue and its replacement by fat (x40). (c) Trephine biopsy showing island of preserved cellularity ('hot spot', x40).

Figure 13.1 Aplastic anaemia. (a) Bone marrow aspirate showing fragments with typical lacy appearance and fatty hypocellular trails (x80). (b) Bone marrow trephine biopsy showing absence of haemopoietic tissue and its replacement by fat (x40). (c) Trephine biopsy showing island of preserved cellularity ('hot spot', x40).

in chromosome breakages and aberrations characteristic of Fanconi anaemia, an inherited from of aplastic anaemia (see Chapter 12).

Paroxysmal nocturnal haemoglobinuria (PNH) should be excluded by performing a Ham test and/or flow cytometry. Analysis of phosphatidylinositol glycan (PIG)-anchored proteins such as CD55 and CD59 by flow cytometry is the much more sensitive test for PNH. Small PNH clones occur in around 25% of patients with aplastic anaemia, but the clinical significance of a small PNH clone is 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, which will be detected by the Ham test. Urine should also be examined for haemo-siderin 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 (see Chapter 11).

Special tests may be of interest in the diagnosis and prognosis of aplastic anaemia. Short-term clonogenic cultures of bone marrow show a uniform reduction in aplastic anaemia in both committed and multipotent progenitor cells. The more primitive long-term culture initiating cells are markedly reduced or absent. Stroma from aplastic marrow may be difficult to grow to confluence in vitro, but aplastic stroma is able to support haemopoiesis from normal stem cells but normal stroma is unable to reverse the defect in colony formation from AA stem cells.

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