Immunofluorescence microscopy of the blood smear in myeloproliferative neoplasms

Introduction Myeloproliferative neoplasms (MPN) are clonal hematologic disorders with variable clinical presentation and prognosis. Thrombotic complications are one of the most frequent clinical manifestations. Early diagnosis can be challenging. We previously identified morphological alterations detectable by immunofluorescence microscopy on peripheral blood smears – specifically, a deficiency of platelet alpha granules, and aggregates of the cytoskeletal protein non-muscular myosin IIA (NMMIIA) in red blood cells – in a proportion of MPN patients. This study (DRKS-ID: DRKS00032588) aimed to confirm these findings in an independent cohort and evaluate the efficacy of immunofluorescence microscopy for distinguishing MPN patients from healthy controls based solely on morphological parameters [ 1 ] [ 2 ]. Method Standard blood smears from 147 MPN patients and 99 healthy controls, matched for age and sex, were stained with primary and secondary antibodies and assessed by immunofluorescence microscopy. Two independent, blinded observers evaluated the smears. Subject identity as well as MPN subgroup characteristics was concealed until study completion. Results Among 147 MPN patients, 65 (44%) had polycythemia vera, 27 (18%) essential thrombocythemia, and 46 (32%) primary myelofibrosis. NMMIIA aggregates in red blood cells were observed in 70% of MPN patients (103/147), and co-occurred with diminished platelet alpha granule expression in 55% of cases. An additional 8% (12/147) exhibited isolated alpha granule deficiency. In contrast, NMMIIA aggregates were detected in only 6% of healthy controls, and alpha granule deficiency in 4%. Immunofluorescence microscopy demonstrated a sensitivity of 70.1% (95% CI: 62.7-77.5%) for detecting MPN based on NMMIIA aggregates alone, and a specificity of 95.9% (95% CI: 88.2-98.7%) for detecting MPN based on alpha granule deficiency alone. Specificity rose to 100% when considering co-occurrence of both changes. Conclusion Deficiency of platelet alpha granules and NMMIIA aggregates in red blood cells are characteristic features of MPN patients. Immunofluorescence microscopy assessment of these changes may serve as a valuable supplementary diagnostic approach to decide, whether suspected patients – for instance with unprovoked thrombosis – should undergo detailed diagnostic workup for MPN ([ Table. 1 ]). Table 1 Estimation of diagnostic performance of immunofluorescence in recognizing MPN patients based on the detection of single and combined morphologic changes. Morphological changes Disorder of alpha granules alone NMMIIA in the erythrocytes alone Disorder of alpha granules and NMMIIA aggregates in the erythrocytes Sensitivity 46.9% (95% CI: 36.6-54.9%) 70.1% (95% CI: 62.7-77.5%) 38.8% (95% CI: 30,6-47%) Specificity 95.9% (95% CI: 88.2-98.7%) 93.9% (95% CI: 88.6-98.2%) 100% (95% CI: 100-100%) Positive predictive value 94.5% (95% CI: 88.6-99.1%) 94.5% (95% CI: 88.6-99.1%) 100% (95% CI: 100-100%) Negative predictive value 54.9% (95% CI: 44.5-65.3%) 67.9% (95% CI: 58.4-79.2%) 52.4% (95% CI: 42-63%) Accuracy 66.7% (95% CI: 56.5-77%) 79% (95% CI: 69- 89%) 63.4% (95% CI: 53,1-73.7%) Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany