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
- CD59 Antigens metabolism MeSH
- Cell Differentiation MeSH
- Antigens, CD blood physiology MeSH
- Diagnosis, Differential MeSH
- Erythrocytes metabolism pathology MeSH
- Glycophorins metabolism MeSH
- Immunophenotyping instrumentation methods standards MeSH
- Cohort Studies MeSH
- Leukocytes pathology MeSH
- Humans MeSH
- Monocytes metabolism pathology MeSH
- Neutrophils metabolism pathology MeSH
- Hemoglobinuria, Paroxysmal blood classification diagnosis pathology MeSH
- Leukocyte Count instrumentation methods MeSH
- Flow Cytometry instrumentation methods standards MeSH
- Receptors, Transferrin blood physiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
This commentary discusses particularities of application of the EuroFlow standardization of flow cytometric analyses on three different flow cytometers. The EuroFlow consortium developed a fully standardized approach for flow cytometric immunophenotyping of hematological malignancies and primary immunodeficiencies. Standardized instrument setup is an essential part of EuroFlow standardization. Initially, the EuroFlow Consortium developed and optimized a step-by-step standard operating procedure (SOP) to setup 8-color BD FACSCanto II flow cytometer (Canto), with the later inclusion of Navios (Beckman Coulter) and BD FACSLyric (Lyric). Those SOPs were developed to enable standardized and fully comparable fluorescence measurements in the three flow cytometers. In Canto and Navios, mean fluorescence intensity (MFI) of a reference peak of Rainbow beads calibration particles is used to set up photomultiplier (PMT) voltages for each detector channel in individual instruments to reach the same MFI across distinct instruments. In turn, a new feature of Lyric instruments allows to share collection of attributes that are used to place the positive population at the same position among instruments in the form of assays, as one of its components integrated in the Cytometer Setup and Tracking (CS&T) module. The EuroFlow Lyric assays thus allow for standardized acquisition of 8-color EuroFlow panels on Lyric without the need to setup the PMT voltages on the individual instruments manually. In summary, the standardized instrument setup developed by EuroFlow enables cross-platform inter- and intra-laboratory standardization of flow cytometric measurements. This commentary provides a perspective on the modifications of the standardized EuroFlow instrument setup of Canto, Navios and Lyric instruments that are described in detail in individual instrument-specfic SOPs available at the EuroFlow website.
- MeSH
- Immunophenotyping instrumentation standards MeSH
- Humans MeSH
- Flow Cytometry instrumentation standards MeSH
- Reference Standards MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
A critical component of the EuroFlow standardization of leukemia/lymphoma immunophenotyping is instrument setup. Initially, the EuroFlow consortium developed a step-by-step standard operating protocol for instrument setup of ≥8-color flow cytometers that were available in 2006, when the EuroFlow activities started. Currently, there are 14 instruments from 9 manufacturers capable of 3-laser excitation and ≥8 color measurements. The specific adaptations required in the instrument set-up to enable them to acquire the standardized 8-color EuroFlow protocols are described here. Overall, all 14 instruments can be fitted with similar violet, blue and red lasers for simultaneous measurements of ≥8 fluorescent dyes. Since individual instruments differ both on their dynamic range (scale) and emission filters, it is not accurate to simply recalculate the target values to different scale, but adjustment of PMT voltages to a given emission filter and fluorochrome, is essential. For this purpose, EuroFlow has developed an approach using Type IIB (spectrally matching) particles to set-up standardized and fully comparable fluorescence measurements, in instruments from different manufacturers, as demonstrated here for the FACSCanto II, and Navios and MACSQuant flow cytometers. Data acquired after such adjustment on any of the tested cytometry platforms could be fully superimposed and therefore analyzed together. The proposed approach can be used to derive target values for any combination of spectrally distinct fluorochromes and any distinct emission filter of any new flow cytometry platform, which enables the measurement of the 8-color EuroFlow panels in a standardized way, by creating superimposable datafiles.
- MeSH
- Hematologic Neoplasms diagnosis MeSH
- Immunophenotyping instrumentation standards MeSH
- Humans MeSH
- Flow Cytometry instrumentation standards MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cerebrospinal fluid (CSF) samples (n=50) from patients with neurological disease (bacterial infection, viral infection, neuroborreliosis and multiple sclerosis) were analysed to characterize cell populations by fluorescent immunocytometry with the CD-Sapphire haematology analyser. Reagent combinations applied to all CSF samples comprised CD3/CD19/HLA-DR and CD4/CD8, with some being further analysed using CD3/CD4, CD3/CD16 and CD3/CD25 protocols. Of the 50 samples, 11 were excluded because of high proportions of nonviable cells (n=2) or insufficient cell numbers (n=9). Apart from bacterial infection with granulocytosis, all diagnostic groups showed high proportions (51.4-77.0%) of CD3+ T cells. There was a modest association between T-cell and B-cell counts, but absolute B-cell numbers exceeded 5 cells/microl in only 7/39 cases (neuroborreliosis, n=6; bacterial meningitis, n=1). CD3/Ia antigen (activation) co-expression was low and only exceeded 5% in 7/39 samples with no diagnostic correlation. Primary CD4+ and CD8+ T-cell subsets showed similar quantitative trends and CD4/CD8 co-analysis revealed the presence in all diagnostic groups (neuroborreliosis and multiple sclerosis in particular) of a CD4+CD8int fraction that was predominantly CD3+ and CD16- and had a morphological profile consistent with small lymphoid cells. Supplementary CD-Sapphire cellular immunological analysis of most CSF samples is feasible using the procedure detailed in this communication.
V současné době dochází k rychlému nárůstu nových poznatků v biomedicíně, který je mimo jiné umožněn neustálým vývojem kvantitativních a kvalitativních analytických metod. Tyto nové přístupy umožňují podrobné sledování dějů na buněčné a molekulární úrovni, a tak nacházejí své uplatnění v široké škále vědních oborů i klinické praxi. Závěry jimi získané jsou přínosem pro civilní i vojenskou medicínu. Jednou z progresivních metod, která dosáhla v posledních letech výrazného technologického rozvoje, je metoda průtokové cytometrie (flow cytometry, FCM). Ve své podstatě FCM představuje dnes již standardní přístup pro vysokorychlostní rozměrovou a typovou analýzu suspenzí částic, nejčastěji savčích buněk. Článek se zabývá principem a nejběžnějšími aplikacemi FCM.
Permanent development of qualitative and quantitative analytical methods has currently resulted in a fast increase of knowledge in biomedicine. Such new approaches have enabled very detailed observations of processes on the cellular and molecular level and thus become useful in both, research and clinical practice including military medicine. Flow cytometry (FCM) is one of the highly progressive techniques with considerable technological development. Nowadays FCM represents a standard approach for a high speed analysis of particle (mostly mammalian cells) suspensions in terms of size, internal complexity and subset composition. The article deals with principle and the most common FCM applications.
- MeSH
- Biomedical Technology methods trends MeSH
- Financing, Organized MeSH
- Fluorescent Antibody Technique methods instrumentation utilization MeSH
- Immunophenotyping methods instrumentation trends MeSH
- Leukocytes immunology classification MeSH
- Lymphocytes immunology classification MeSH
- Antibodies, Monoclonal classification therapeutic use MeSH
- Polymerase Chain Reaction methods trends utilization MeSH
- Flow Cytometry methods instrumentation utilization MeSH
