FLOW CYTOMETRIC CLASSIFICATION OF 350 TYPE III PNH CLONES IN ITALY: A MULTI-CENTRIC STUDY

Background: So far , three different types of PNH have been described, based upon the evidence of bone marrow failure (BMF) and hemolysis: a) florid PNH (hemolysis+/BMF-), b) PNH within a BMF syndrome (hemolysis+/BMF+), c) subclinical PNH (hemolysis-/BMF+). In any case, the presence of a PNH clone detected by Flow Cytometry is a prerequisite for PNH diagnosis. In 2010, an Italian archive of FCM-detected PNH clones (HYPERLINK “http://int.clonepnh. com/” http://int.clonepnh.com/) was created on a multi-laboratory basis. Aims: We had two main goals: a) to provide a large dictionary of Italian PNH clones; b) based upon stringent rules regarding the compilation of records, to obtain an auto-educational effect on participating laboratories. Methods: Ninety-one laboratories participated in the project. We analyzed data from ClonePNH Archive to evaluate: a) how many PNH clones have been identified so far and how they are classifiable on the basis of the Reason for Testing (RFT) reported in clinical request; b) how many clones received more than one FCM determinations; c) among these, which was the initial RFT as well as the fate of the clone. Data refer to analysis registered in ClonePNH Archive up to 02/20/2013. Results: Here, we describe the cellular composition and the clonal evolution of 350 type III (complete defect of GPI-linked proteins) PNH clones identified during the study. Forty-nine of these clones (16.6%) were accompanied by a PNH-II clone (partial defect of GPI-linked proteins). Hemoglobinuria was the most frequent (45.4%) reason for testing (RFT), followed by aplastic anemia (AA, 17.7%), MDS (11.1%), unexplained cytopenia (UC, 8%), hemolytic anemia (5.7%), , BMF (4.6%), atypical venous thrombosis (3.1%), other (8%). Fluorescent Aerolysin (FLAER) was used since 2007, with an increasing % of utilization, from 4% to 60% of cases. CD24 utilization also progressively increased. CD59 was the most used antigen for RBC typing. The most used gating strategies were based upon physical parameters for RBC, CD45 and/or CD33 vs side scatter for granulocytes and monocytes. The 350 clones were categorized into 3 classes according to their size, determined as the percent of PNH cells in peripheral granulocytes: 0.01-10% (134 clones, or 38.3%, defined as “small”), 10.1%>70% (74 clones, or 21.1%, defined as “intermediate”), 70.1%>100% (131 clones, or 37.4%, defined as “large”). This distribution was significantly different from that expected on the basis of a random distribution within the three classes (i.e. 10%, 60% and 30%): chi square was 51 with a p value <0.0001. Ninety-six clones were sequentially studied (with a follow up ranging from 3 to 74 months): Twenty-nine of them (30%) showed a change in category (19 increased and 10 decreased). Just 5 clones jumped from “smallest” to “biggest” category or vice versa (three increased ant 2 decreased). Summary / Conclusion: This is the first multi-laboratory relational database of FCM-detected PNH clones described so far. An auto-educational goal was reached, since general sensitivity increased progressively and reagent choice significantly changed, leading to a stable FCM protocol, consisting of FLAER and CD24 for granulocytes, FLAER and CD14 for monocytes, CD59 for erythrocytes. As regards clonal evolution, the rarity of migration between extreme categories suggests that the belonging to these ones could be sustained by different backgrounds and pressures,as suggested by different association with HLA genotypes.