Role of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Refractory Anemias

The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in the setting of aplastic anemia (AA) and myelodysplastic syndrome (MDS) highlights the pathogenetic link between these disorders. The awareness that small clones of PNH cells could be detected by flow cytometry in aplastic anaemia patients who had negative Ham test has led to classifying PNH into two broad groups such as Haemolytic PNH which can have 50% presenting with thrombosis and a hypo plastic group which behaves like aplastic anaemia. Even if there is a small PNH clone in the setting of aplastic anaemia, then the patients respond better to immunosuppressive therapy.

Flowcytometry is a relatively new advanced technique in many parts of India and there is no widespread consensus among methodology. Further, the clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is yet to be established here.

To validate the role of flowcytometry in the diagnosis of PNH, in the setting of aplastic anaemia, myelodysplastic syndrome or abdominal or cerebral vein thrombosis in the setting of PNH which may help to prognosticate the course and treatment.

The study was both retrospective and prospective. The prospective arm of the study was conducted from September 2009 to April 2010. Ham's test and Sucrose lysis test were done as screening tests and were reported as positive or negative. Other laboratory studies including hemogram and bone marrow analysis were done as part of standard care. In the retrospective group there were 32 patients who were diagnosed as PNH based on Ham's and Sucrose lysis test followed up from 1990.

Four color flowcytometry immunophenotypic analysis with CD55 and CD59 was used to detect PNH clones in RBCs. EDTA-anticoagulated whole blood was stained with anti–CD 55-Phycoerythrin (PE) and anti–CD 59-PE.A minimum of 10,000 events were acquired. For analysis, RBCs were identified by light-scatter properties. For the purpose of the study, 3% of type II and/or type III cells were considered as threshold to diagnose PNH clones in both CD55 and CD 59 gated population. The results of laboratory studies including transfusion history and flow cytometry were correlated. Institutional ethical committee approval was obtained for the conduct of the study.

33 patients were included as part of the prospective group. The number of males was more compared to females (19:14). 78% of patients had hemoglobin of less than 7gm%. The presentation varied from unexplained anemia to thrombosis. Based on flow cytometry, 3 patients were found to have PNH clones in the RBC lineage by both CD 55 and CD 59. CD 55 alone picked up one more patient. In the retrospective group there were 32 patients who were followed up from 1990. Interestingly, two of them who had earlier presented with aplastic anemia had transformed into PNH in a span of 3years and 6 months. All patients were treated with Stanazolol and cyclosporine. The median survival was 12.14. months with Kaplan-Meier survival estimates of 30 and 20 percent at 121 months, and 143 months after diagnosis, respectively. The patients in the prospective group are being followed up.

The Ham test, sucrose lysis test, and modified Ham tests rely on the differential sensitivity of PNH red cells to haemolysis. These tests, although suitable for haemolytic PNH, cannot reliably detect small populations of PNH red cells nor differentiate partially and completely deficient cells. One patient who presented with DVT in whom increase in clone size was demonstrated by serial flowcytometry. Testing for the presence of PNH RBC clones provides additional diagnostic information and the thrombotic event may probably directly related to the size of the PNH clone.

Conclusions: Our data demonstrate the utility of RBC assay in flow cytometry in the primary screening of PNH, AA, and MDS patients. The clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is relatively new and could be demonstrated in one patient.

No relevant conflicts of interest to declare.