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
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematopoietic stem cell disorder resulting from the somatic mutation of the X-linked phosphatidyl-inositol glycan complementation Class A (PIG-A) gene. Depending on the severity of the mutation in the PIG-A gene, there is a partial or absolute inability to make glycosylphosphatidyl-inositol (GPI)-anchored proteins including complement-defense structures such as CD55 and CD59 on RBCs and WBCs. Flow cytometric detection of PNH clones has become the gold standard and has played an increasingly important role in the diagnosis, monitoring, and clinical management of patients with PNH. Recently, a 4-part set of Consensus Guidelines have been published by flow experts in the field to address the key assay-specific considerations for the identification of PNH clones in RBC and WBC, how to report such data and a full validation document for the assays described. Below, we have summarized the most significant aspects of this International effort.
- MeSH
- antigeny CD55 krev genetika MeSH
- antigeny CD59 krev genetika MeSH
- konsensus MeSH
- lidé MeSH
- membránové proteiny krev genetika MeSH
- paroxysmální hemoglobinurie krev diagnóza genetika MeSH
- průtoková cytometrie metody normy MeSH
- směrnice pro lékařskou praxi jako téma MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: CD157 has been recently reported as a useful glycosylphosphatidylinositol (GPI)-linked marker for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with suspected paroxysmal nocturnal hemoglobinuria by flow cytometry as it targets both neutrophils and monocytes. The aim of this study is to test the feasibility of a non-fluorescent aerolysin (FLAER) approach and propose an alternative for laboratories, where FLAER is not available. METHODS: We validated a non-FLAER-based single-tube, 6-color assay targeting the GPI-linked structures CD157, CD24, and CD14. We determined its performance characteristics on 20 PNH patient samples containing a variety of clone sizes and compared results with a previously validated FLAER-based approach. RESULTS: Coefficient of variation (CV) for intra-/interassay precision analyses ranged from 0.1%/0.2% to 3.02%/7.58% for neutrophils and from 0.10%/0.3% to 5.39%/6.36% for monocytes. Coefficient of determination (r2 ) for linear regression analysis of PNH clones from 20 patients ranging from 0.06% to 99.7% was 0.99 in all cases, Wilcoxon ranks test showed no statistically significant differences (P > 0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from 0.06 to 0.2. CONCLUSION: Our results confirm very good performance characteristics for both intra- and interassay precision analyses, favorable correlation, and agreement between the FLAER and non-FLAER-based approaches, using the CD157 GPI marker. Our experience suggests that a rapid and cost-effective simultaneous evaluation of PNH neutrophils and monocytes by flow cytometry without using FLAER is possible in areas where FLAER may not be widely available. © 2016 International Clinical Cytometry Society.
- MeSH
- ADP-ribosylcyklasa imunologie metabolismus MeSH
- antigen CD24 imunologie metabolismus MeSH
- antigeny CD14 imunologie metabolismus MeSH
- bakteriální toxiny MeSH
- biologické markery metabolismus MeSH
- CD antigeny imunologie metabolismus MeSH
- cytotoxické proteiny tvořící póry MeSH
- GPI-vázané proteiny imunologie metabolismus MeSH
- lidé MeSH
- monocyty imunologie metabolismus MeSH
- neutrofily imunologie metabolismus MeSH
- paroxysmální hemoglobinurie imunologie metabolismus MeSH
- průtoková cytometrie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články 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.
