Detection of small populations of CD59-deficient erythrocytes in patients with aplastic anemia or myelodysplastic syndrome and normal individuals.

To detect a small population of blood cells with a deficiency of glycosyl phosphatidylinositol (GPI)-anchored protein, we evaluated the expression of CD59 by flow cytometry on one million erythrocytes, which is about 100 times more than the number of erythrocytes tested by our standard immunoassay. Blood samples from healthy volunteers, patients with aplastic anemia (AA), and patients with myelodysplastic syndrome (MDS), who all showed no detectable GPI deficiency by the standard assay, were investigated. The numbers of CD59-deficient erythrocytes were 5 to 145/10(6) erythrocytes in the healthy volunteers (mean 29.2), and one of the volunteers had an increase in the deficient cells exceeding the mean + 3 SD (141.7), a normal limit. A CD59-deficient population was detected in 6 of the 21 (28.6%) patients with AA and 5 of the 18 (27.8%) patients with MDS. The new assay was performed again in 5 of these 11 patients and the normal individual who had the CD59-deficient populations at 6 and 12 months after the initial study. The number of deficient cells gradually increased in 1 patient with MDS (from 511 to 2892/10(6) erythrocytes), while the numbers of the other 4 patients showed a tendency to decline, although the deficient populations were repeatedly detected on most of the occasions. Changes in the number of the deficient cells were also seen in the healthy volunteer, but they were rather rapid; the numbers changed from 145 to 5661 and then to 18/10(6) erythrocytes within 3 months. The CD59 assay used in this study is easy to perform and enabled us to detect less than 1% GPI-deficient cells.

CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia.

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG-A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG-A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG-A gene were all different and included two single-base insertions, one single-base deletion, two two-base deletions, and one each of eight-base insertion and nine- and ten-base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI-anchoring defect and that the PIG-A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG-A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.

[LGL lymphoma].

We reported in this article two patients with large granular lymphocytic lymphoma (abbreviated as LGL lymphoma). One was the patient with LGL leukemia/lymphoma (patient 1) and other was the patient with NK-LGL lymphoma (patient 2). Because the gene of TCR delta was rearranged in the patient 1, the clonality of the LGL leukemia/lymphoma was confirmed. However, it is not determined yet whether the lineage of tumor cells is T cells or NK cells. The cytochemical features of the lymphoma cells of the patient 2 were studied. It was found that NK cell-derived lymphoma cells of the patient were positively stained with these two monoclonal antibodies that are reactive with T cell; one is anti-CD45RO (UCHL-1) and other is anti-CD3. Judging from the result, malignant NK-LGL cells in some patients are cytoplasmic CD3+ and UCHl-1+. It is emphasized that May-Grünwald-Giemsa stain of biopsied specimen of the lymphoma is required for making the diagnosis of LGL lymphoma.

Analysis of PIG-A gene in a patient who developed reciprocal translocation of chromosome 12 and paroxysmal nocturnal hemoglobinuria during follow-up of aplastic anemia.

The relationships between paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and myelodysplastic syndrome (MDS) are not clear. Here we describe a patient, J20, who developed a reciprocal translocation of chromosome 12 and PNH during follow-up of AA. All metaphases in CD59-deficient bone marrow mononuclear cells had the translocation, whereas none of the CD59-deficient cells had it, indicating that the PNH clone coincided with a cell population bearing the chromosomal aberration. We found a somatic single-base deletion mutation in the PIG-A gene of this patient's peripheral blood cells. This is the first patient with PNH with a PNH clone containing a chromosomal translocation.