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- Material and methods
Because of the insensitivity of the Ham test, paroxysmal nocturnal haemoglobinuria (PNH) has been inaccurately viewed as a late clonal complication of aplastic anaemia (AA). To clarify the relationship between PNH and marrow failure, we tested for the presence of glycosylphosphatidyl-anchored protein-deficient (GPI-AP) granulocytes in large cohorts of patients with AA, myelodysplasia (MDS), and pure haemolytic PNH. A PNH clone was detected in 32% of new AA patients and 18% of MDS patients. In serial studies, this proportion did not change up to 15 years after diagnosis, suggesting that expansion of aberrant cells is an early event (i.e. prior to initial presentation). For all patients with a PNH clone, on average 14% of PNH granulocytes were found on presentation and 37% at 10 years. Patients with PNH but without cytopenia showed higher percentages of GPI-AP-deficient cells than did those with the AA/PNH syndrome. After immunosuppression, there was no change in the contribution of PNH clone to blood production, arguing against the ‘immune-escape’ theory in PNH. Clinically, a high proportion of GPI-AP-deficient cells correlated with marrow hypercellularity. GPI-AP-deficient cells were similarly present in patients with and without karyotypic abnormalities. Our results indicate that the GPI-AP-deficient clones show quantitative and kinetic differences between classic haemolytic PNH and PNH with marrow failure, in which the evolution rate is low later in the course of the disease.
The relationship between aplastic anaemia (AA) and paroxysmal nocturnal haemoglobinuria (PNH) has been known for years (Dacie & Lewis, 1961; Dameshek, 1967; Dacie & Lewis, 1972; Young, 1992; Luzzatto et al, 1997; Young & Maciejewski, 1997; Hillmen & Richards, 2000; Young & Maciejewski, 2000) but because of the insensitivity of the Ham test, traditionally used for the diagnosis of PNH, it has been believed that this rare disease is a late clonal complication of AA or even related to the use of immunosuppression (Socie et al, 2000). Flow cytometric diagnosis of PNH has altered the view on the association between these two diseases (Schrezenmeier et al, 1995,2000; Dunn et al, 1999). The presence of PNH clones has been found more often in AA patients than was estimated from the results of the Ham test (Dunn et al, 1999; Richards et al, 2000) but the availability of sensitive tests has raised even more questions as to the nature of the pathophysiological relationship between these two diseases (Luzzatto et al, 1997; Hillmen & Richards, 2000; Young & Maciejewski, 2000).
Although its diagnosis is invariably linked to detection of deficiency in membrane expression of glycophosphoinositol-anchored proteins (GPI-AP), PNH is a clinically heterogenous disease and can overlap with AA in the PNH–AA syndrome and with myelodysplasia (MDS; Iwanaga et al, 1998; Dunn et al, 1999), as well as occurring in a ‘pure’ haemolytic form without clinical signs of deficient blood cell production or disturbed marrow morphology. Various permutations of the clinical course of PNH are possible including development of pancytopenia after the diagnosis of haemolytic disease or, conversely, evolution of PNH from a typical AA.
Here, we used a flow cytometry-based test to detect the presence of PNH clones. Owing to several factors (transfusions, destruction of GPI-AP-deficient erythrocytes and transfer of GPI-AP such as CD55 and CD59 to negative erythrocytes), detection of a PNH clone in granulocytes is more accurate then erythrocyte-based assays (Dunn et al, 1999; Piedras & Lopez-Karpovitch, 2000; Richards et al, 2000). Using this precise method, we designed an analytic approach to PNH in which, in addition to categorizing patients according to their primary pathological diagnosis, we studied a cohort of patients selected solely by a singular parameter, the presence on GPI-AP-deficient clone. Using this criterion, we attempted to establish on clinical grounds the pathophysiological relationship between a PNH clone its size and manifestations of marrow failure. In contrast to many other studies that provide a ‘snap shot’ analysis of the frequency of PNH clone in AA, we serially studied a large number of patients with AA for the presence of GPI-AP-deficient clone.
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- Material and methods
Clarification of the relationship between AA, an immune-mediated bone marrow failure, and PNH, the result of expansion of a somatically mutated haematopoietic stem cell, has eluded investigators for several decades (Dacie & Lewis, 1961; Dameshek, 1967; Dacie & Lewis, 1972; Young, 1992). In this study, we attempted to characterize the clinical significance of PNH clonal expansion in a large population of patients suffering from a variety of haematological diseases. In contrast to many early studies, which were based on the insensitive Ham test, we used a flow cytometric assay that is both specific and sensitive. Using the presence of GPI-AP-deficient granulocytes as a basis, we studied two parameters: the relative clone size in individual patients and the frequency of detection of an expanded PNH clone in various patient populations.
In choosing a threshold level of > 1% granulocytes lacking two GPI-AP, we recognized the discordance between this value and the mean ± 2SD (95% confidence interval) in our large number of controls. The more conservative, higher figure was chosen to avoid false positives because we did not observe a distinct population of double-negative cells below the 1% level. Nevertheless, we cannot exclude that such cells in controls and in patients represent minimal expansion of a minor PNH population and not a technical artefact. AA patients, who were considered negative for the presence of PNH clone, showed higher values within the population of GPI-AP-deficient granulocytes, suggesting that low levels of PNH cells may be present even more frequently in AA than we have determined. The presence of PIA-G mutation in small numbers of cells has been also demonstrated in normal individuals (Araten et al, 1999). Other investigators have used very small numbers of GPI-AP-deficient erythrocytes and granulocytes to conclude that very high proportions of AA patients have concurrent PNH (Brodsky et al, 2000) but, clearly, selection of the indicator proteins may affect results. We used two GPI-AP that are constitutively expressed on granulocytes; others have chosen CD55 or CD59 (Piedras & Lopez-Karpovitch, 2000; Richards et al, 2000; Schrezenmeier et al, 2000). Our selection was based on direct experimental comparisons, as well as the fact that CD16 and CD66b are constitutively expressed on normal granulocytes but not on erythocytes or platelets. It is also possible that some abundant GPI-AP, such as CD55 and CD59, are transferred in vivo from positive to negative cells (Sloand et al, 1998), which would lead to a decrease in the sensitivity of PNH detection.
In the patient populations tested, small size PNH clones were most common. The size of abnormally expanded clones did not fit a normal distribution, suggesting that the initial appearance of a PNH clone was not a single, stochastically determined event. Greatly expanded clones correlated well with the presence of clinical haemolysis. However, despite measurement on granulocytes, there was little correlation between the detection of GPI-AP-deficient cells and the degree of cytopenia. Furthermore, significant expansion of PNH clones was observed despite persistent or recurrent cytopenias, from which we infer that evolution of PNH does not always coincide with haematopoietic recovery or reflect escape from immune attack. Although institutional referral bias may play a role in the interpretation of our data, patients with a history of AA were those most frequently observed with expanded PNH clones, and even symptomatic haemolysis was more often associated with AA than occurred as typical primary haemolytic PNH. Pure haemolytic PNH was less frequent as a clinical manifestation of a large PNH clone, and it may represent the extreme pole of a broad clinical spectrum of possible PNH manifestations. Although some AA patients showed greatly expanded PNH clones at first clinical presentation, in many others expansion was associated with the development of a more cellular bone marrow and haematological improvement, consistent with compensatory repopulation or escape. There was no relationship between an expanded PNH clone and the development of clonal karyotypic abnormalities. Expanded PNH clones were found in all age groups, and there was no correlation between patient age and the size of the clone.
Like others, we found a high incidence of expanded PNH clones in patients with AA (Schrezenmeier et al, 1995; Azenishi et al, 1999; Dunn et al, 1999). We also systematically analysed large numbers of patients over time. Assuming a constant rate of evolution of PNH, a steady increase in the frequency of detection of the PNH clone as patients are observed would be expected. However, in our series, almost all PNH was discovered at the time of diagnosis of aplasia, suggesting that the evolution of PNH may be linked to the initiating events of AA. Expansion of a PNH clone correlated with higher neutrophil counts and increased marrow cellularity. In addition, a highly expanded PNH clone was more frequently found in patients with a long history of disease compared with the low values seen in most of the patients at presentation. Nevertheless, in most patients, PNH clone size did not change in a consistent pattern as a result of immunosuppressive therapy or relapse. If PNH evolution was as a result of escape from immune system attack, as has been proposed (Rotoli & Luzzatto, 1989; Young, 1992), response to immunosuppression should result in a decrease in the relative size of the GPI-AP-deficient clone. However, immune pathophysiology of PNH clone was suggested by the relationship between the presence of the GPI-AP-deficient clone and response to immunosuppression.
In MDS, a much lower proportion of patients showed an expanded PNH clone and in some patients with the MDS/PNH syndrome, AA preceded the development of secondary MDS. Nevertheless, PNH in association with typical MDS (Iwanaga et al, 1998; Dunn et al, 1999) suggests a close pathophysiological relationship between MDS and AA (Barrett & Saunthararajah, 2000).
The high proportion of AA patients with concurrent PNH at diagnosis demands explanation, and further laboratory investigations are required to clarify the operation of possible mechanisms including secondary immune escape of PNH clone or its primary involvement in the induction of the immunological process leading to AA.