A-152 Enhancing PNH Diagnosis with Multicolor Flow Cytometry Immunophenotyping Protocol in a Medical Center in Taiwan
Authors: Fang C , Liao Y , Yang S , Wen Y , Lin C 
Published: October 2024

Abstract

Background
Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from mutations in the X-linked PIG-A gene, leading to an inability to produce GPI-anchored proteins on RBCs and WBCs. PNH presents in three variants: classic (hemolytic), associated with specified bone marrow disorders, and subclinical. Flow cytometric analysis stands as the gold standard for PNH detection, crucial in diagnosis, monitoring, and clinical management. Recognizing potential variations in PNH testing among labs, it is essential to establish a precise and consistent protocol for accurate diagnosis and patient management. This report aims to demonstrate the effectiveness of a PNH immunophenotyping protocol adhering to ICCS/ESCCA PNH consensus guidelines.

Methods
A total of 501 samples were analyzed from Jan 1st, 2021 to Dec 31st, 2023. All the acquisitions were accomplished by a 3-laser and 10-PMT Navios EX (Beckman Coulter) flow cytometry. A typical combination of anti-CD235a-FITC (clone KC16) and anti-CD59-PE (clone MEM43) antibodies were adopted to identify the PNH clone size in the RBC tube. To achieve higher sensitivities in PNH granulocyte and PNH monocyte detection, a single tube, 6-color mixture of FLAER, monoclonal antibodies including CD14-APC, CD24-PE, CD33-APC-H7, CD15-PacB, and CD45-KrO, to immunostaining simultaneously was developed. An unstained control was run after the full-stained tube to determine the position of threshold for positivity, and to exam the potential carryover into the test sample.

Results
RBC and granulocyte assays can detect PNH phenotypes at the 0.009% level or higher with more than 50,000 cells of gated cells acquired, while the monocyte assay can detect at &gt;=0.069% with at least 20,000 cells acquired. Analysis was conducted on five hundred and one patients to investigate potential PNH clones in their blood cells. Among them, 45 patients were identified as having classic PNH, while PNH clones were not detected in 168 cases, constituting 33.5% of total cases. Minor PNH clone (&lt;1.0%) in single cell lineage were observed in 144 cases, making up 28.7% of all cases. Minor PNH clones found in more than one cell lineages were found in 155 cases (30.9% of 501). Additionally, minor PNH clones were identified across RBCs, granulocytes, and monocytes, with percentages of 32.9%, 23.8%, and 43.9%, respectively of all patients. Notably, monocyte clones exceeded granulocyte clones in 23.0% of cases with WBC PNH clones, irrespective of clone sizes. Furthermore, we investigated seven patients exhibiting persistent minor PNH clones in 2 to 3 cell lineages. Among them, five had aplastic anemia or myelodysplastic syndromes, while one underwent evaluation for young stroke and one was investigated for venous thromboembolism.

Conclusions
Flow cytometry labs aim not only to identify classic PNH patients but also to consistently detect minor or small clones in individuals with bone marrow disorders or subclinical conditions. Our findings are compatible with international reports, underscoring the importance of routinely examining patients with minor clones. Such assays serve as essential tools for ongoing monitoring, facilitating PNH diagnosis and management by physicians.