BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.
- MeSH
- antigeny CD59 metabolismus MeSH
- buněčná diferenciace MeSH
- CD antigeny krev fyziologie MeSH
- diferenciální diagnóza MeSH
- erytrocyty metabolismus patologie MeSH
- glykoforin metabolismus MeSH
- imunofenotypizace přístrojové vybavení metody normy MeSH
- kohortové studie MeSH
- leukocyty patologie MeSH
- lidé MeSH
- monocyty metabolismus patologie MeSH
- neutrofily metabolismus patologie MeSH
- paroxysmální hemoglobinurie krev klasifikace diagnóza patologie MeSH
- počet leukocytů přístrojové vybavení metody MeSH
- průtoková cytometrie přístrojové vybavení metody normy MeSH
- receptory transferinu krev fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. © 2017 International Clinical Cytometry Society.
- MeSH
- erytrocyty metabolismus MeSH
- konsensus MeSH
- leukocyty metabolismus MeSH
- lidé MeSH
- monocyty metabolismus MeSH
- neutrofily metabolismus MeSH
- paroxysmální hemoglobinurie diagnóza metabolismus MeSH
- počet leukocytů metody MeSH
- průtoková cytometrie normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. METHODS: We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. RESULTS/CONCLUSIONS: CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2 = 1) and monocytes (R2 = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.