Nachweis von Aggregaten aus nicht-muskulärem-Myosin IIA in Erythrozyten mittels Durchflusszytometrie
Martina Wolff (Greifswald / DE), Leonard Swensson (Greifswald / DE), Jan Wesche (Greifswald / DE), Julia Klauke (Greifswald / DE), Thomas Thiele (Greifswald / DE), Tina Schnöder (Hannover / DE), Florian Heidel (Hannover / DE), Andreas Greinacher (Greifswald / DE)
Myeloproliferative neoplasms (MPN) are hematologic disorders characterized by clonal proliferation of one or more blood cell lines. Some MPN are associated with mutations in the JAK2 gene and affected patients have an increased thromboembolic risk. We could recently show, that patients with MPN show aggregates of the cytoskeletal protein non-muscle myosin IIA (NMMIIA) in erythrocytes in immunofluorescence microscopy (IF). we developed a flow cytometry-based method to detect NMMIIA aggregates in erythrocytes for early detection of MPN.
Flow cytometry (FC) method was established using a mouse model and further validated with human samples. Whole blood was drawn from of Jak2+/VF mice (mutant) and corresponding Jak2+/+ wildtype mice (WT); and from patients with genetically confirmed MPN and healthy controls. Blood samples were fixed, permeabilized and stained with anti-NMMIIA-AF647 and anti-235a-PE. AF647-baseline fluorescence was set using samples of wild type mouse or healthy controls without NMMIIA-aggregates. We simultaneously prepared blood smears from human samples, fixed, and stained them with primary anti-NMMIIA- and secondary AF568-labelled antibody.
Jak2-mutant mice showed a higher proportion of NMMIIA-aggregates compared to WT-mice in flow cytometry (8.6 % vs 1.3 %, p=0.0014). In patients with MPN and visible NMMIIA aggregates in IF, the mean percentage of NMMIIA-positive events in FC was 11.1 % vs 0.48 % in healthy controls (p<0.0001). MPN-patients with no visible NMMIIA aggregates in IF displayed similar results in FC as healthy controls (0.66 %, p= 0.1422). Human blood samples were stored in glycine buffered saline for 7 days and analyzed again. Results were comparable to fresh samples. (healthy control 0.43%, MPN-patient with aggregates 14.6 %; MPN-patient without aggregates 0.73 %). None of the 50 healthy controls showed NMMIIA-aggregates in IF. 10 patients had aggregates in IF, of which only 1 was negative in FC
Aggregates of NMMIIA in erythrocytes can be detected by flow cytometry. The assay is fast and easy to apply to screen patients with possible MPN. We hypothesize that NMMIIA-aggregates in erythrocytes may also be found in patients with clonal hematopoiesis (CH), a stem cell aging related phenomenon, clinically ranging from healthy individuals to progression into MPN. This is currently investigated in an ongoing study.
Disclosures available upon request