Detection of Paroxysmal Nocturnal Hemoglobinuria Clones in Patients with Idiopathic Venous Thromboembolism

Background. Paroxsymal Nocturnal Hemoglobinuria (PNH) is an uncommon disease with an estimated population prevalence of 0.002%, however the estimated prevalence of venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) or pulmonary embolism (PE), in PNH patients is 25 to 33%. Small PNH clones have been identified in patients with unexplained splanchnic vein thrombosis and in people with apparently normal blood counts and might predispose these patients to thrombosis. Furthermore, only about 40-50% of idiopathic VTE patients are affected by one or more thrombophilias. The presence of PNH clones as a contributing factor to a hypercoagulable state has never been examined in this population.

Patients and Methods. In order to determine the prevalence of PNH clones in patients with prevalent idiopathic DVT/PE we conducted a cross-sectional study at the Thrombosis Clinic of the London Health Sciences Centre in London, Ontario, Canada, between May 2011 and June 2014. We included patients with at least one episode of objectively demonstrated DVT or PE according to previously published criteria. Patients were excluded if they had: a) a previous diagnosis of PNH, b) well established predisposing risk factors for VTE in the 3 months preceding the VTE, c) active cancer other than non-melanoma skin cancer within 6 months of VTE, or if they were unable to provide written informed consent. Clinical and laboratory information was abstracted from the patient charts. Patients were screened for PNH using a high sensitivity flow cytometric assay which can detect at least 0.01% GPI deficient erythrocytes using CD235a and CD59 in a 2 color assay. Additionally, a modification of our previously developed fluorescent aerolysin (FLAER) assay was used to detect GPI deficient neutrophils using FLAER, the GPI linked protein CD24, CD45 and CD15 (a mature neutrophil marker) in a 4 color flow cytometric assay. A control group of 30 normal donors was used to standardize, determine and validate the level of sensitivity of both the red and white cell assays. The primary outcome of the study was the presence of a PNH clone >0.02% in erythrocytes or neutrophils. Secondary outcome was the presence a PNH clone of any size. Assuming an underlying PNH proportion in the population of 0.002%, we estimated that a sample size of 402 patients achieved 80% power to detect a proportion of 0.4%. Confidence intervals for proportions were calculated using the Wilson score method.

Results

We included 394 patients of which 388 had samples available for flow cytometry. The characteristics of the included patients are shown in Table 1. One patient (0.26%; 95% CI 0.05 to 1.45) had a detectable PNH clone in the neutrophil population (0.02%) whereas another patient had a detectable PNH clone below the positivity threshold (<0.01%). Neither patient had evidence of hemolysis or cytopenias.

Conclusion

Very small PNH clones can be detected in a small proportion of patients with unexplained VTE but without clinical manifestations of hemolytic PNH. Their relation with the development of VTE is yet to be determined.

n, Number of patients with risk factor; N, number of patients with valid information;

SD standard deviation; CI confidence interval