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.
Subjects:
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.
Results:
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%.
Conclusion:
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.
