Paroxysmal Nocturnal Haemoglobinuria Clone In Aplastic Anaemia Patients At The Beginning Of Disease and In Remission
Authors: Fidarova Zalina , Mikhailova Elena , Lugovskaia Svetlana , Naumova Elena , Troitskaia Vera , Ustinova Elena , Pochtar Margarita , Daria Kisilichina , Galtseva Irina , Shitareva Irina , Kuzmina Larisa , Parovichnikova Elena , Savchenko Valeriy 
Published: October 2019

Abstract

Introduction
Aplastic Anaemia (AA) and Paroxysmal Nocturnal Haemoglobinuria (PNH) are severe hematological diseases accompanied by bone marrow failure syndromes. The high-sensitivity flow cytometry standartised methods helped us to detect the PNH clone incedence at AA patients at the different stages of disease and of treatement and to reveal its’ influence on the immunosuppressive therapy (IST) effectiveness.

Objective
to detect the PNH clone at AA patients at different stages of disease and to reveal its’ influence on the IST effectiveness.

Methods
63 patients with severe AA (SAA) who received combined IST with antithymocytic globulin (hATG) and cyclosporin A (CsA) have been included into the study. Mediane age – 26 years (16-65).
All 63 patients were divided into 2 groups. The 1st one included de novo AA patients (n=28); the 2nd group – AA patients in complete remission (CR) after IST (n=35). The median remission duration was 3 years (2-6 y). The results of the de novo AA treatment (1stgroup) were evaluated at 3, 6 and 12 months from the start of IST.
We used the flow cytometry (Becton Dickinson (BD) FACS Canto II and Beckman Coulter (BC) FC 500) to evaluate the PNH clone.
Peripheral blood samples were analyzed with antibodies CD45(BD), CD15(BD), CD64(BD), CD235a(BC), GPI-tying antibodies CD59 (Invitrogen), CD14(BC), CD24(BC) and FLAER (Cedarlane). Minor PNH clone was detected when the count of GPI- deficient cells did not exceed 1%.

Results
The PNH clone was found in 18 patients among 28 (64%) from the 1st group. The minor clone was found in 4 patients, in 3 patients the clone size exceeded 50%. Median (Me) clone size on the Red Blood Cells (RBC: type II + type III) was 0,25% (0,03-25,3%), Granulocytes (GR) – 1,7% (0,02- 93,92%), Monocytes (Mon)- 23,2% (0,05-95,66%).
7/18 SAA patients (38,9%) with the PNH clone, showed a haematological response at 3 months from the treatment start, including 3 patients with the minor PNH clone. 2/18 patients (11.1%) underwent allogenic bone marrow transplantation (alloBMT). 9/18 did not get the remission by the 3d month.
4/18 patients (22,2%), without response at 6 months, received the second course of IST; 5/18 patients (27.7%) are being followed up and can‘t be analyzed at 6 months.
It worth to note, that PNH clone disappeared after allo-BMT (n=2) and in 1 patient with CR after IST.
None of 10 patients without PNH clone attained response at 3 months (p=0,02). 4 out of 10 (40%) achieved only partial remission at 6 months. In these cases minor PNH clone appeared after hematological response and persisting from 6 till 18 months. 6 other patients are still on the treatment (ATG).
In the 2nd group the PNH clone was detected in 26 of 35 cases (74,3%), only 11 of them had a minor clone. The Me of PNH clone size on RBC - 1,4% (0,02 to 3,76%), Gr – 25,2% (0,01-93,73%), Mon - 23,52% (0,01-54,32%)

Conclusion
The PNH clone has been detected in more than 60 % of de novo SAA patients. The disease was characterized by pancytopenia and aplasia of the bone marrow without clinical signs of intravascular hemolysis. In our study we observed the quick IST response at 3 months in SAA patients with the PNH clone (38,9%), in patients without PNH clone at time of diagnosis achievement of partial remission at 6 months was followed by PNH clone appearance and persistence.

Disclosures:
No relevant conflicts of interest to declare.