The Frequency of Red Cells with a Spontaneously Appearing XK-Null (McLeod-like) Phenotype Is Normal In Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Abstract 816

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a large population of blood cells with an acquired somatic mutation in the X-linked PIG-A gene. This results in an inability to synthesize glycosylphosphatidylinositol (GPI) and an inability to express on the cell surface any of the proteins that require this structure. Although there are case reports of progression to leukemia or high grade myelodysplasia, in large series of patients, this occurs only rarely. It is therefore hypothesized that in PNH, the large GPI (-) population results from selection in favor of a mutant stem cell clone in an aplastic marrow environment, without inherent predisposition to mutations. In favor of this hypothesis is the observation that the GPI(-) population is often found to have a single PIG-A mutation. An alternative hypothesis, however, is that there may be inherent hypermutability in patients with PNH; indeed, it is not unusual that 2 or even several PIG-A mutant clones can be shown to coexist in the same patient by careful sequence analysis, and cytogenetic abnormalities are not uncommon in PNH. We have previously reported a normal rate of spontaneously arising GPI (-) cells among established GPI (+) B lymphocyte lines from patients with PNH, arguing against this model, but this does not rule out the possibility that hypermutability somehow spares the lymphoid lineage. We have therefore now analyzed red blood cells from patients with PNH to determine the frequency of spontaneously arising cells with a mutation in another gene, XK, which is associated with McLeod syndrome. Like PIG-A, the XK gene resides on the X-chromosome, a large spectrum of mutations can inactivate the gene, and its associated mutant phenotype is detected by flow cytometry—as a loss of Kell antigen expression on red cells. As for PIG-A, we have previously demonstrated that healthy individuals harbor very small populations of blood cells with a spontaneously appearing XK-null (McLeod-like) phenotype, in this case at a frequency of about 39 × 10⁻⁶ in healthy adults and 9 × 10⁻⁶ in cord blood samples. In order to determine the frequency of McLeod-like cells, approximately 3 ul of whole blood is first incubated on ice with a hybridoma supernatant (MIMA 91) which has specificity for a common non-polymorphic Kell antigen. The cells are then washed twice again and stained with a secondary PE-conjugated rabbit anti-mouse antibody, washed twice again, and then stained with a FITC-conjugated anti-glycophorin A (GPA) antibody, and then washed again. At least 2 million GPA (+) events are then collected on a FACScan instrument. Stored cells from patients with the McLeod syndrome and obligate heterozygote carrier females serve as a control. In general, spontaneously appearing McLeod-like cells in normal donors form a distinct population that is strongly GPA (+) and dim for Kell antigens, typically having <20% of the mean PE fluorescence of the overall population. Here we have analyzed samples from 17 patients with PNH, 13 of whom would be classified as “classic PNH”. Among the 17 patients, the median blood counts were 2100/ul for the ANC, 10.8 gm/dl for the HGB, 138,000/ul for the platelet count, and 125,000/ul for the absolute reticulocyte count. The median % PNH clone size was 31% for the RBC and 77% for the PMN's. 7 of the patients had a history of Aplastic Anemia, 6 had a history of thrombosis, and 10 were receiving Eculizumab infusions. We have shown that among these 17 patients, the median frequency of spontaneously arising McLeod-like red cells was 46 × 10⁻⁶ (range 14 to 204 × 10⁻⁶). In comparison, among 15 normal healthy adults, the median frequency of McLeod-like cells was 48 × 10⁻⁶ (range 14 to 113 × 10⁻⁶), whereas the median frequency was 15 × 10⁻⁶ (range 11 to 37 × 10⁻⁶) among 5 cord blood samples. We conclude that there is no evidence for hypermutability in stem cells or in progenitors of the red cell lineage among patients with PNH using the XK gene as a sentinel for spontaneous somatic mutations.