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Cytometry B Clin Cytom. 2015 Jul-Aug;88(4):261-9. doi: 10.1002/cyto.b.21225. Epub 2015 Feb 3.

Rare event counting of CD59- red cells in human blood: A 47-month experience using PNH consensus guidelines for WBC and RBC testing in a reference lab.

Author information

1
Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, Utah, 84108.
2
Division of Hematology, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, Utah, 84132.
3
Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah, 84132.

Abstract

INTRODUCTION:

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder characterized by increased complement-mediated lysis of erythrocytes (RBCs) because of low/absent glycophosphatidylinositol (GPI) anchors of numerous cell surface proteins.

METHODS:

Rare event analysis was applied to 120 million RBCs (12 normal individuals) and 102 million RBCs (102 normal individuals) to establish a reference range and verify a methodology for rare event analysis. Patient PNH testing (n = 10,984) was performed over 47 months using the 2010 consensus guidelines with CD59-PE and glycophorin A-FITC for RBCs and FLAER-Alexa 488, CD33-PerCP-Cy5.5, CD15-APC, CD14-APC-Cy7, and CD24-PE for WBCs.

RESULTS:

The distribution of CD59- RBCs in the normal population was asymmetric with a mean of 5.9, median between 3 and 4, and mode of 2 per million RBCs. The normal range of CD59- RBCs was 0-17/million RBCs. A natural cutoff of 2.5% of the peak expression of CD59 delineates CD59+ from CD59- populations. The incidence of GPI- samples received by the laboratory was 6-7% without correlation to age (P = 0.35) or sex (P = 0.45). The percentage of GPI- neutrophils and monocytes were strongly correlated (R(2) = 0.96) and usually greater than the percentage of GPI- RBCs.

CONCLUSION:

PNH RBC testing is a good example of rare event analysis applied to clinical cytometry—only 2.5 min are required to collect 1 million RBCs. With an established normal range of CD59- RBCs, the correlation between total cell count and sensitivity for detecting an abnormal population can be calculated using Poisson statistics.

KEYWORDS:

CD59; FLAER; PNH; glycophorin a

PMID:
25605539
DOI:
10.1002/cyto.b.21225
[Indexed for MEDLINE]
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