A Franco-Belgian Workshop on PNH Diagnosis by Flow Cytometry: Results of a Multicentric Evaluation of Technical Aspects and Standardized Procedures of Reference Assays on Behalf of the French Association of Cytometry (AFC)

Abstract 4370

In 2010, technical guidelines have been published in order to standardize the diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH) by multiparameter flow cytometry. General recommendations have been released including the use of a new reagent, the FLAER and different monoclonal antibodies (MoAb).

To make an inventory of the practices and to evaluate the contribution of these new guidelines in routine diagnosis, we have conducted a multicentric study in 39 French and Belgian laboratories comparing three different strategies. EDTA blood samples from 141 healthy controls, 80 myelodysplasia (MDS), 83 myelocytosis and 76 previously diagnosed PNH were analysed. We have strived to address the question of sensitivity and defined a preanalytic approach, using red blood cell lysis prior to staining, to allow the recording of a high number of polymorphonuclear leucocytes (PMN >200 000). We applied two published techniques (i) Nakao’s strategy, a two-color staining (CD55/CD59 versus CD11b) (ii) the North American guidelines through a multicolor panel (five or six colors including FLAER and MoAb) and (iii) a strategy including MoAb (workshop combination) without FLAER. Reagents were provided by the companies. Analysis were performed on 3 different flow cytometers from Beckman Coulter (FC500: n=17, Navios, n=9) and Becton Dickinson (Canto II: n=13).

Whatever the strategy used and irrespective of the flow cytometer, our results showed that each center was able to reach a sensitivity of 1%. When considering PMN, a 0.1% threshold was even achievable in most cases after the first round of interpretation. The results on monocyte population were poorer due to the low number of cell recorded. As our aim was to improve the ability to detect PNH clones and harmonize the multiparametric techniques, we established a standardized gating procedure using either basophils, lymphocytes or NK cells as internal control cell populations. Then a second round of data interpretation was performed. When considering the healthy control samples, the comparison of the two sets of results showed that there was a striking improvement of the cellular background after applying the standardized gating procedure on PMN population. A 1 to 2-log decrease was observed with the 6-color combinations. Therefore we refined the performance of the technique by establishing the limit of detection (LOD), defined as the mean cellular background + 2 standard deviations, for each technique and staining combinations. Using the standardized procedure, we found that LOD determined on healthy controls samples with more than 10⁵ PMN were: 0.0054%, 0.0024% on the 6 color cytometers Navios and Canto, 0.092% on the 5 color cytometer FC500 with combination (ii) and 0.0072%, 0.0044% and 0.206% respectively with combination (iii). As myelocytosis and MDS have a higher cellular background after the first round of interpretation, the improvement is more significant when samples were tested using the optimized procedure. Thus, we observed that, compared to healthy controls, the LOD determined on myelocytosis and MDS samples was not modified in the series processed on Canto ((ii) 0.0048% and 0.0019%, respectively, (iii) 0.0066% and 0.0031%) whereas it was mildly increased in Navios series ((ii) 0.0594% and 0.0106%, (iii) 0.0148% and0.0246%) and in FC500 series ((ii) 0.1837% and 0.7120%, (iii) 0.7557% and 0.5862%). Finally, the procedure was successfully validated on PNH samples since we observed no obvious modifications of the proportion of the clone after applying the gating.

In conclusion, this study shows that the detection of large clones can be achieved with all approaches, the detection of small clones requiring standardized preanalytic, analytic and interpretation procedures. This study highlights the contribution of the use of control cell populations in the gating strategy and supports the use of FLAER. Moreover, an optimized procedure appears to reach a very good LOD of 10⁻⁴. Finally, there is a need for the implementation of quality controls including pathological samples to ensure technical validation and QC monitoring.