[1444] High Sensitivity PNH Testing: The Reference Lab Experience

Dragan Jevremovic, Simon D Althoff, Michael M Timm, William G Morice, Curtis A Hanson. Mayo Clinic, Rochester, MN

Background: Flow cytometry immunophenotyping (FCIP) of peripheral blood cells for expression of GPI-linked proteins is the test of choice for the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). As new technologies are becoming available, higher sensitivity testing is increasingly expected as the standard of practice.
Design: Following guidelines published by the International PNH Interest Group, we have developed a high-sensitivity PNH assay by FCIP. The assay was implemented in February 2010 and offered as a reference laboratory test. Results from a four month period in 2011 were compared with the results from the same period in 2009.
Results: In the high sensitivity assay a minimum of 150,000 RBCs and WBCs each are collected. The RBC portion of the test utilizes antibodies to CD45 and CD235a for gating, and CD59 for the assessment of type I, II, and III RBCs. The WBC portion of the test is a 8-color single tube using antibodies to CD14, CD15, CD16, CD24, CD33, CD45 and FLAER. Test validation which included 120 normal specimens established detection sensitivities of 0.01% for type III RBCs and granulocytes; 0.05% for monocytes; 1% for type II RBCs.
In the period of March-June 2009, our laboratory analyzed 1147 peripheral bloods for the presence of a PNH clone using an assay collecting 10,000 cells, expression of CD59, CD14, and FLAER. The sensitivity of this assay had been validated at 3%. Out of 1147 analyzed cases, 24 were found PNH-positive in both RBCs and WBCs, and 8 in WBCs only (total 32; 2.8%). In 2011, during the same time period, 1118 cases were analyzed by the new high-sensitivity method; of these, there were 55 PNH-positive cases in both RBCs and WBCs, and 23 in WBCs only (total 78; 7.0%).
Conclusions: By introducing a new high-sensitivity test for PNH we have increased by 2.5 times the rate of detection of PNH clones. Furthermore, clones present in WBCs only (more likely to be of a small size) were detected 3 times as frequently with the high sensitivity test. No difference in the distribution of test orders is seen, so the difference in the rate of detection is attributable to the higher sensitivity testing only. These results show that the rate of detection of PNH clones is significantly increased by the implementation of high sensitivity flow cytometry. As International PNH Interest Group guidelines recommend regular follow-up for all positive clones, this finding has a significant impact on patient care, both in terms of quality of life and health care costs.
Category: Hematopathology

Wednesday, March 21, 2012 9:30 AM

Poster Session V # 211, Wednesday Morning


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