As is often the case of many other rare diseases, PNH incidence has been poorly reported with just a few published registry studies, none of them covering Polish population. Based on the available data, PNH incidence is generally estimated at 1–1.8 cases per million individuals worldwide uniformly affecting population with different genetic backgrounds [2, 10, 11]. Extrapolating these values, in Polish population of 37.75 million, a range of 38–68 new cases could be expected annually. In the studied period, a total of 322 patients with a median of 31 cases a year with PNH clone of any size (95% CI 25–39) and 19 cases a year with PNH clone > 1% (95% CI 14–23) were diagnosed in our laboratory, that theoretically comprise 45.6–81% of the predicted cases. Despite the single-center nature of our data, that comprise major limitation of the study, we believe that this dataset can be considered representative for Polish population.

The annual number of diagnosed cases did not differ significantly within the studied period and remained at the stable level with a single outlying value in 2020, when COVID-19 pandemic exerted the most pronounced influence on healthcare systems worldwide. In that period, although the number of referred patients did not differ from median for the studied period (n = 284 vs median of 288, 95% CI 218–331), the number of those who tested positive for PNH clone (n = 24 vs median of 31, 95% CI 25–39, and 8.45% vs median of 10.63%, 95% CI 9.67–13.61) and the number of patients with PNH clone > 1% (n = 11 vs median of 19, 95% CI 14–23, and 3.9% vs median of 6.7, 95% CI 5.1–9.4) were significantly lower than the observed median (Fig. 2BC). Numerous studies have confirmed that due to pandemics, the access to healthcare services in 2020, including care provided by hematology centers in Poland, was markedly limited, resulting in decreased number of hospitalizations and delayed diagnoses made in the affected period [12, 13]. Contrary to this trend, lower PNH clone detection rate in total and more frequent detection of small clones observed in our cohort, did not result from decreased number of referrals, as this value remained at the expected level. Recent studies have shown that the incidence of idiopathic AA continuously declined from the beginning of the pandemics until the end of 2020 [14] that may explain decreased PNH clone detection rate in our cohort, especially that AA patients constituted the majority of the PNH-positive patients in the study group. The changes in hematology practice in the country or diagnostic access bias (preferential access to services for cytopenic and MDS patients with small clones) cannot be excluded. Detailed analysis of this phenomenon is beyond the scope of this study; however, it might be speculated, that the reduced PNH clone detection rate might be caused by factors directly or indirectly related to COVID pandemics.

In our cohort, 322/2790 (11.5%) new cases of PNH clone were detected, including 40.6% of cases (128/322) with very small clone or rare PNH cells (≤ 1%) (Fig. 2BC). Similar detection rate was observed in Spain (10%) [15], while other centers reported much higher (16% in Brazil [15]) or lower (6.48% in Dahl‐Chase Dx Services, Bangor, US [8]) rates that may be related to local clinical practice and varying indications for screening. Similarly to our observations, very small clones, detected with high-sensitive protocols, were reported for 44% cases in the US cohort [8] confirming the benefits of high-sensitive testing in the PNH screening, especially in bone marrow failure patients who tend to have lower clone sizes.

The most common indication for PNH screening in the published studies are AA and other cytopenias, constituting 34.5%-65.6% of cases, followed by hemolytic anemia as a second commonest indication observed in 19%-30.7% of cases [15,16,17]. In our cohort, AA and cytopenias were much more common, constituting 76% of cases, while hemolysis was relatively rare indication for screening constituting 8% of cases (Fig. 3). This data suggests a significant underrepresentation of patients with hemolytic anemia in the population screened for PNH clone in routine clinical practice in Poland resulting in relative overrepresentation of AA and cytopenia patients. In our experience, bone marrow failure patients are treated in specialized hematology centers that strictly follow the current guidelines and refer most of these patients to PNH screening [7]. Hemolytic anemia patients, on the other hand, are more often treated outside of tertiary reference hematology centers, and therefore may be not referred for PNH screening as a part of differential diagnosis. Pure thrombotic presentation of PNH was also less common in our cohort in comparison to published data (1.2% vs 2.1% or 22.1% in UK [18] and Belgian [17] cohorts, respectively); however, the differences between reported cohorts are too large to draw any conclusions.

According to the expert consensus on PNH diagnosis, at least 2 cell lineages need to be tested to confirm PNH diagnosis [4]. However, as highlighted in the same guidelines, evaluation of the PNH clone in all three lineages is becoming the standard of care for PNH-suspected cases. As the presented dataset has been collected in real-life clinical setting for a decade, and most of the material has been studied based on previous diagnostic guidelines, most of the patients evaluated in his paper has been tested in two lineages only.

High-sensitivity quality of the assay has been assured by stringent verification and validation of the assay according to the standards set up by the consensus guidelines [4, 8]. As this is a real-life, clinical laboratory dataset, no additional antibody specificities nor additional staining, such as live/dead counterstaining, have been added to the antibody cocktails. Although some authors suggest that the addition of 7AAD or DRAQ7 counterstaining may improve the quality of the data obtained and reduce non-specific reagent binding positively influencing the evaluation of the PNH clone size [19, 20], we decided not to modify the established staining methods described in the current guidelines.

As observed in previous studies [21, 22], in most patients clone sizes in neutrophil and monocyte lineages are closely correlated (Fig. 4D). Interestingly, in a few cases in our cohort with rare PNH cells or very small clone size in neutrophils (n = 14, 14.9% of patients with both clones measured), clone sizes exceeding 1% were observed in monocyte lineage highlighting the need to evaluate both leukocyte lineages to achieve comprehensive clone size evaluation (Fig. 4B). Also, median clone size was uniformly higher for monocytes than any other cell lineage tested in all subpopulations (Fig. 4C). We cannot exclude, that this phenomenon may have occur due to different sensitivity of neutrophils and monocytes to cell death, as cell death markers counterstaining was not used [19, 20]. However, in our hands and as described in the current guidelines [4, 8], the sensitivity of PNH clone detection is high even in the absence of counterstaining. Moreover, the difference between monocyte and neutrophil clones was visible regardless of the origin of the sample (samples from our center tested a few hours after blood drawing and samples from other centers tested with a delay allowing for transport).

As expected, clone size of PNH RBCs, due to RBC susceptibility to complement-mediated hemolysis and clone size changes due to blood transfusions, correlate weakly or do not correlate with the size of PNH clones in leukocyte lineages, especially in cases with large RBC clones (Fig. 4D). For the same reason, typical bimodal distribution of clone size [3] was observed for leukocyte lineages, but not for RBCs (Fig. 4A). Noteworthy, the peak for the largest clones in the histogram (Fig. 4A) was lower in comparison to the data published so far confirming underrepresentation of hemolytic patients in our cohort [3, 21].

The major limitations of our study were single-center nature and lack of extensive clinical data. However, although the presented data has been collected in a single laboratory, the studied patients were referred for testing from 35 hematology centers from the entire country that extends the geographical range of the studied population much beyond our region, making the obtained data representative for the country population. On the other hand, the fact that the studies were performed in a single unique cytometry unit offers additional advantage of better standardization of the results obtained. Since a significant number of patients in our cohort were referred to our laboratory for testing only, in these cases data on indication for screening was obtained from referral documents only without chart review, that may have affected data completeness (e.g. due to underreporting of thrombosis or hemolysis in bone marrow failure patients). In this line, as the data on blood transfusion was not available for all patients, we decided not to include this confounding factor that may strongly affect PNH clone size in RBC. In addition, although only patients who tested positive for the presence of PNH cells in two lineages, the choice of the lineage tested was at referring physician discretion. In consequence, data for monocytes and RBCs are not available for all patients that may limit statistical power of some evaluations.

To conclude, we present the largest cohort of patients diagnosed with PNH clones/ cells in real-life routine clinical practice in Poland published so far. Our data confirms the efficiency of high-sensitivity testing recommended by International Clinical Cytometry Society Consensus Guidelines, especially in patients with bone marrow failure. Although our findings are mostly consistent with the available body of data from other countries, we observed marked underrepresentation of hemolytic patients in PNH screened population and PNH-positive cases. Contrary to the expectations, classic PNH was one of the rarest indications for PNH screening, suggesting that in routine practice in Poland differential diagnosis of hemolytic anemia rarely include PNH. This highlights the need for improved education of clinical community about PNH and indications for PNH clone screening. To better understand epidemiology of PNH and patients’ characteristics, initiation of national registry of patients burdened with PNH clone is warranted.