2010 年 20 巻 1 号 p. 65-74
High-sensitivity analysis of erythrocytes using flow cytometry (FCM) is performed for a definitive diagnosis of subclinical paroxysmal nocturnal hemoglobinuria (PNH).
In this study, we conducted line-specific analyses of CD55/59 antigens using FCM by CD235a/CD11b gating of erythrocytes/granulocytes, compared the results between these two cell fractions, and evaluated the causes of differences.
Peripheral blood samples collected in EDTA from 88 subjects were employed. Three, 43, 24, and 18 of them had PNH (including 1 case of PNH/AA), aplastic anemia, myelodysplastic syndrome (MDS), and other disorders, respectively.
Analyses of erythrocytes and granulocytes showed that the rate of concordance was 72%. All subjects with PNH, 47% of AA cases, and 4% of MDS cases were PNH-cell-positive in both erythrocytes and granulocytes. Differences in the results between the two cell fractions were observed in 22 cases (25%), and, in most cases, the PNH cells were positive only in erythrocytes. This discrepancy was noted in 23, 25, and 33% of AA, MDS, and other disorders, respectively, showing similar rates in all disorders. This occurred around the regions of cells showing a low expression of CD235a in scattergrams.
Since the involvement of small-sized erythrocytes with a smaller surface area of the cell membrane was suspected as the cause for the difference, CD55/59 fluorescence intensity depending on the size of erythrocytes was compared.
It was revealed that the expression of CD55/59 decreased with the size of erythrocytes, and that most PNH cells were small erythrocytes.
On the other hand, when only larger-sized erythrocytes were analyzed, the concordance rate with granulocytes rose to 88%.
With high-sensitivity analysis of PNH erythrocytes, small-sized erythrocytes with a low expression of CD55/59 are likely to be recognized as PNH blood cells, which causes false-positive results.