pnh flow cytometry test procedure

The flow cytometer machine then sorts the cells by type and color. CD16a: Responsible for ADCC and involved in phagocytosis. Flow cytometry (FCM) is the method by which the various characteristics of individual particle or cells are studied. Anti-CD71 is useful to distinguish mature RBC from immature RBC (reticulocytes and nucleated RBC). Lab technicians or pathologists perform flow cytometry. Hill A., Kelly R.J., Hillmen P. Thrombosis in paroxysmal nocturnal hemoglobinuria. This can help to determine the cause of anemia. DeZern A.E., Brodsky R.A. Paroxysmal nocturnal hemoglobinuria: a complement-mediated hemolytic anemia. Evaluation and comparison of different approaches for the detection of PNH clones by flow cytometry following the ICCS guidelines. Deficiency of lymphocyte function-associated antigen 3 (LFA-3) in paroxysmal nocturnal hemoglobinuria. Our website is not intended to be a substitute for professional medical advice, diagnosis, or treatment. Unless your healthcare provider gives you specific instructions, there is usually no need for preparation before having a flow cytometry analysis. Anemia happens when your bone marrow cant make enough red blood cells to compensate for the blood cells destroyed by your complement system. (https://pubmed.ncbi.nlm.nih.gov/32357555/), (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879566/), (https://www.ncbi.nlm.nih.gov/books/NBK562292/). Kinoshita T., Ohishi K., Takeda J. GPI-anchor synthesis in mammalian cells: genes, their products, and a deficiency. sickled cells). Red blood cells were stained with the following combinations of mAbs: anti-CD59-FITC (clone MEM-43; Invitrogen), anti-CD55-PE (clone IA10; Becton Dickinson), anti-CD235a-APC (clone GA-R2/HIR2; BD Biosciences), and anti-CD45-KO (clone J.33; Immunotech). Cell type-specific expression of two genes that differ in single nucleotide substitutions. F1000Research 2016; 5(F1000 Faculty Rev): 209. The 8-color tube that has been used for PNH screening in WBC in our Laboratory, which combines non-GPI-linked (CD10, CD45, CD64) with FLAER and GPI-AP (CD14, CD16, CD66b, CD157) also allows to detect PNH populations at the 0.01% level (Fig. PNH testing is recommended for patients with intravascular hemolysis, acquired bone marrow failure syndromes and thrombosis with unusual features. Other laboratory tests are recommended in patients suspected of having PNH, to document the existence and severity of the hemolytic anemia and/or the associated cytopenias, as well as to exclude other possible causes for the anemia and other cytopenias. AA or MDS) had large PNH populations in granulocytes (>50%) and only a minority (8%) had small populations (<10%). (iii), Use monoclonal antibodies against CD45 (for WBC) and at least 1 non-GPI-AP lineage specific marker to better identify each cell population, Use appropriated gating strategies for each blood cell population. As so, mAbs against lineage-specific integral membrane proteins have been used for lineage identification (e.g., CD15 and/or CD33 for neutrophils; CD33 and/or CD64 for monocytes). We do not endorse non-Cleveland Clinic products or services. Epub 2017 Sep 8. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, clonal hematopoietic stem cell disorder, characterized by a somatic mutation in the gene encoding phosphatidylinositol glycan class A ( PIGA) gene 1. The test requires a sample of blood, bone marrow, tissue or other body fluid. The novel monoclonal antibody By114 helps detect the early emergence of a paroxysmal nocturnal hemoglobinuria clone in aplastic anemia. Flow cytometry is a laser-based technique used to detect and analyze the chemical and physical characteristics of cells or particles. Besides CD55 and CD59, many other GPI-AP are expressed in blood cells, where they function as enzymes, receptors and adhesion molecules, being also involved in signal transduction (Table1). Deep next-generation sequencing on all exons of known genes of the GPI anchor pathway in PNH patients without somatic PIGA mutations allowed for the identification of mutations in PIGT, an autosomal gene (20q13) that is essential for attachment of the preassembled GPI anchor to proteins [130]. Fortunately, these side effects wear off soon after people start treatment. An abnormal cell will show different patterns that may suggest the presence of leukemia, lymphoma or other diseases. Fang X., Zheng P., Tang J., Liu Y. CD24: from A to Z. Elishmereni M., Levi-Schaffer F. CD48: a co-stimulatory receptor of immunity. During the process, a sample of cells or particles is suspended in fluid and injected into a flow cytometer machine. PMC Flow cytometry: Surface markers and beyond. PNH by Flow Cytometry | Laboratory Test Guide | Dept. of Laboratory Bn M.C., Castoldi G., Knapp W., Rigolin G.M., Escribano L., Lemez P., Ludwig W.-D., Matutes E., Orfao A., Lanza F., vant Veer M. EGIL, European Group on Immunological Classification of Leukemias CD87 (urokinase-type plasminogen activator receptor), function and pathology in hematological disorders: a review. Rotoli B., Luzzatto L. Paroxysmal nocturnal hemoglobinuria. Richards S.J., Hill A., Hillmen P. Recent advances in the diagnosis, monitoring, and management of patients with paroxysmal nocturnal hemoglobinuria. This is generally done on a bone marrow sample. FSC and SSC, represented as FSC integral and SSC peak, were captured on a linear scale; for fluorescence parameters, a logarithmic amplification was used. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder named for a single symptom: Red/brown/dark urine noticed during late night or early morning trips to the bathroom. PNH clone assessment by flow cytometry and its clinical - Springer Following the current recommendations and participating in the above mentioned EQA/PT programs made possible to perform PNH testing by FCM according to the good laboratory practices. Flow cytometry testing in PNH. how much is enough? Schrezenmeier H., Hertenstein B., Wagner B., Raghavachar A., Heimpel H. Apathogenetic link between aplastic anemia and paroxysmal nocturnal hemoglobinuria is suggested by a high frequency of aplastic anemia patients with a deficiency of phosphatidylinositol glycan anchored proteins. Funaro A., Ortolan E., Ferranti B., Gargiulo L., Notaro R., Luzzatto L., Malavasi F. CD157 is an important mediator of neutrophil adhesion and migration. For instance, neutrophils and monocytes may be contaminated with eosinophils and dendritic cells, respectively. Overview of Flow Cytometry and Microbiology. This is accomplished by processing cells through a common flow cytometry protocol. and transmitted securely. Mortazavi Y., Merk B., McIntosh J., Marsh J.C.W., Schrezenmeier H., Rutherford T.R. By Elizabeth Boskey, PhD JMH blood group system: a review. Epidemiology. CD16b, the GPI-anchored form, is expressed mainly in neutrophils, is involved in neutrophil activation and induction of an adhesive phenotype and has two polymorphisms (NA-1 and NA-2). Paroxysmal Nocturnal Hemoglobinuria (PNH), High Sensitivity, RBC and Laboratory studies for paroxysmal nocturnal hemoglobinuria, with This is generally done on a blood sample using flow cytometry analysis. In PNH, a gene called PIGA in one stem cell mutates or changes into an abnormal stem cell. According to the UK-NEQAS survey mentioned above, the most commonly used reagents to detect GPI-deficient WBC were FLAER (86%) and anti-CD24 (71%) for granulocytes, and FLAER (63%) and anti-CD14 (81%) for monocytes [29], which were the reagents recommended at the time the survey was implemented (2013) [25,27], and still continue to be suggested by the current guidelines [31]. Morado M., Subir D., Lpez Rubio M. Grupo Espaol para el Estudio de Hemoglobinuria Paroxstica Nocturna por Citometra de Flujo, Paroxismal nocturnal hemoglobinuria: new treatments and general guidelines for diagnosis. Lublin D.M., Mallinson G., Poole J., Reid M.E., Thompson E.S., Ferdman B.R., Telen M.J., Anstee D.J., Tanner M.J. Molecular basis of reduced or absent expression of decay-accelerating factor in Cromer blood group phenotypes. Tate C.G., Uchikawa M., Tanner M.J., Judson P.A., Parsons S.F., Mallinson G., Anstee D.J. In addition, the Iberian Society for Cytometry has an EQA scheme for PNH screening based on the analysis of FCM data files, in which several Spanish and Portuguese centers are taking part [51]. Both low and high values can be fine, or problematic, depending on the circumstances. Flow cytometry may be used whenever your healthcare provider needs to learn more about the cells inside your body. The difference is hematuria is red blood cells in your urine, while hemoglobinuria is hemoglobin in your urine. In accordance, the recent guidelines of the British Society for Standards in Haematology recommended that all patients with AA be screened for PNH using FCM, and then reassessed periodically [50]. A semen sample would be expected to take longer, and bone marrow samples will be more complicated samples to take, depending on the sedation you'll need for it. Parker C, Omine M, Richards S, Nishimura J, Bessler M, Ware R, Hillmen P, Luzzatto L, Young N, Kinoshita T, Rosse W, Soci G. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. FCM studies using FLAER and fluorochrome conjugated mAbs against GPI-AP and non-GPI-AP should be considered the appropriate method for high sensitivity tests to detect minor PNH populations, and they are recommended when studying BM failure syndromes, such as AA and MDS [[111], [112], [113], [114]]. Cleveland Clinic Laboratories now offers high sensitivity flow cytometry testing for PNH using whole blood. Use adequate samples and proper immunofluorescence techniques. A weakly supervised deep learning approach for label-free imaging flow-cytometry-based blood diagnostics. -, Luzzatto L. Recent advances in the pathogenesis and treatment of paroxysmal nocturnal hemoglobinuria. 42% of PNH+ patients had a PNH clone of <1%.*. Rnne E., Pappot H., Grndahl-Hansen J., Hyer-Hansen G., Plesner T., Hansen N.E., Dan K. The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria. Ortolan E., Vacca P., Capobianco A., Armando E., Crivellin F., Horenstein A., Malavasi F. CD157, the Janus of CD38 but with a unique personality. Samples submitted outside of this range will be evaluated for quality using appropriate internal controls and the results qualified appropriately. CD38 NADase/ADP-ribosyl cyclase gene family . You also may wish to consider support from people living with cancer, HIV, or a similar diagnosis to your own. This test evaluates bone marrow function and red blood cell production. ii. This type of testing can check the number of immune cells, assess your cell cycle status, identify cancer cells or even analyze your DNA. 2022 Apr 8;14(4):e23965. Paroxysmal nocturnal hemoglobinuria (PNH) is currently diagnosed by flow cytometry; although highly sensitive, its interpretation and reporting appear as critical as its technique. Sutherland D.R., Kuek N., Azcona-Olivera J., Anderson T., Acton E., Barth D., Keeney M. Use of a FLAER-based WBC assay in the primary screening of PNH clones. Binds to extracellular matrix proteins such as fibronectin, fibrinogen, laminin and collagen type I. sharing sensitive information, make sure youre on a federal Bivariate dot plots (Panel A) and band diagrams (Panel B) obtained from FCM analysis of the peripheral blood RBC of a healthy individual and of a patient with classic PNH, using the Infinicyt software, version 1.8.0 (Cytognos, Salamanca, Spain). Talk to your healthcare provider, however, about your specific situation. Hernndez-Campo P.M., Almeida J., Snchez M.L., Malvezzi M., Orfao A. ~ Usually performs equally as; > usually performs better than. Abbreviations: AA, Aplastic anemia; FLAER, fluorochrome-conjugated mutant aerolysin toxin; FSC, forward scatter; GPI-AP, glycosylphosphatidylinositol anchored proteins; MDS, Myelodysplastic syndrome; PNH, Paroxysmal nocturnal hemoglobinuria; RBC, Red blood cells; SSC, side scatter; WBC, White blood cells. Flow cytometry is generally used as follow up testing after a complete blood count (CBC) or white blood cells scan (WBC). This indicates that PNH is a complex disorder orchestrated by many genetic alterations, with some of these mutations probably preceding the acquisition of PIGA mutations, while others occurring later [133]. diagnosis of AA or aplastic crisis). Vortex procedures during the incubation period may help to minimize RBC agglutination and the choice of the fluorochrome may also be relevant. To improve diagnostic criteria for PNH, the normal patterns of expression of GPI-AP on different subsets of PB cells were previously characterized in detail [72]. Kulagin A., Lisukov I., Ivanova M., Golubovskaya I., Kruchkova I., Bondarenko S., Vavilov V., Stancheva N., Babenko E., Sipol A., Pronkina N., Kozlov V., Afanasyev B. Prognostic value of paroxysmal nocturnal haemoglobinuria clone presence in aplastic anaemia patients treated with combined immunosuppression: results of two-centre prospective study. In the first 1600 patients enrolled, the median size of the PNH population in granulocytes was off 68.1% (ranging from 0.01%, the minimum inclusion criterion for enrollment, to 100%), and, as expected, it was larger in patients with classic PNH than in patients with AA-PNH (83% vs. 35%) [13]. Maria Lus Queirs for dedication to this area and for the help with Figures, and to the clinicians that have send their patients for study, thereby contributing to our experience in PNH diagnosis. Most people who have PNH develop anemia that requires additional treatment. Kalina T., Flores-Montero J., Lecrevisse Q., Pedreira C.E., J van der Velden V.H., Novakova M., Mejstrikova E., Hrusak O., Bttcher S., Karsch D., Sdek ., Trinquand A., Boeckx N., Caetano J., Asnafi V., Lucio P., Lima M., Santos A.H., Bonaccorso P., van der Sluijs-Gelling A.J., Langerak A.W., Martin-Ayuso M., Szczepaski T., van Dongen J.J.M., Orfao A. Healthcare providers refer to these proteins as the complement system. Quality assessment program for EuroFlow protocols: summary results of four-year (2010-2013) quality assurance rounds. However, they have no diagnostic advantage compared to PB studies, and should not be used routinely for PNH diagnosis. The assay has been validated for the diagnosis and monitoring of patients with hematopoietic neoplasms. Johnston J.J., Gropman A.L., Sapp J.C., Teer J.K., Martin J.M., Liu C.F., Ye X., Ye Z., Cheng L., Brodsky R.A., Biesecker L.G. Bone marrow cell immunophenotyping has been done in research settings, to better understand the abnormal patterns of expression of GPI-AP observed in maturating hematopoietic cells from PNH patients [79,101,116,117,120], as compared to healthy individuals [73]. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but often debilitating disease which may lead to death in up to 35% of patients within 5 years if unrecognized and untreated. Abbreviations: GPI, glycosylphosphatidylinositol; WBC, white blood cells; PNH, Paroxysmal Nocturnal Hemoglobinuria; RBC, red blood cells. Histocompatibility (HLA) testing is done prior to an organ transplant, whether someone is intended to be a donor or a recipient. Wang D., Fu R., Ruan E.-b., Qu W., Liang Y., Wang H.-q., Wang J., Li L.-j., Liu H., Wang H.-l., Zhang T., Liu H., Wu Y.-h., Xing L.-m., Wang G.-j., Wang X.-m., Song J., Guan J., Shao Z.-h. STAT5 phosphorylation levels of erythropoietin and thrombopoietin receptors in CD34(+)CD59(-) and CD34(+)CD59(+) bone marrow cells of patients with paroxysmal nocturnal hemoglobinuria. Therefore, RBC analysis alone is not recommended (and is not enough) for PNH diagnosis. Diagnosis and potential treatment of blood and bone marrow cancers. Kinoshita T., Medof M.E., Silber R., Nussenzweig V. Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria. For example, genetic deficiency of CD16 determining the presence of CD16 negative neutrophils in the PB may occur, although this is a rare condition [96], and patients with MDS may have abnormal expression of some GPI-AP (e.g., low levels of CD16 expression in neutrophils) [34]. 2014;25(2):194-202. doi:10.1097/EDE.0000000000000043. We do not endorse non-Cleveland Clinic products or services. Then a fluorescent dye is attached to the antibody. Additionally, UW Hematopathology is unable to accept flow cytometry reference testing from new clients and will be limiting service to a subset of current clients. Involved in fibrinolysis, cell adhesion and migration. Induces monocyte activation. When red blood cells fall apart, they dump their hemoglobin into your bloodstream. Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. The site is secure. Gupta R., Pandey P., Choudhry R., Kashyap R., Mehrotra M., Naseem S., Nityanand S. Aprospective comparison of four techniques for diagnosis of paroxysmal nocturnal hemoglobinuria. Involved in complement-mediated cell lysis and ADCC. This cell divides and makes additional abnormal stem cells that become abnormal red blood cells and platelets. Syaloglycoprotein, Mucin-like molecule. Flow cytometry may be recommended if your healthcare provider needs to detect, identify or count specific cells. Moreover, in patients with classic PNH, anemia is frequent and the leukocyte and platelet counts are usually normal or nearly normal, whereas leukopenia and/or thrombocytopenia invariably accompany PNH in the setting of another BM failure syndrome. The test allows healthcare providers to look at specific cells one at a time. WBC analysis by FCM has advantages in PNH population detection and size assessment. Monoclonal antibodies for flow cytometry-based paroxysmal nocturnal hemoglobinuria detection For many years, PNH detection in our FCM laboratory was based on two-color FCM assays using specific combinations of antibody-fluorochrome conjugates ( Chart 1 ). Advertising on our site helps support our mission. Anti-CD235a mAbs are known to cause RBC aggregation, especially if conjugated with phycoerythrin; mAb titration is needed for each batch used. Sutherland D.R., Richards S.J., Ortiz F., Nayyar R., Benko M., Marinov I., Illingworth A. CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria. This will depend on the specific types of cells under investigation, as well as what lab is used. Pakdeesuwan K., Wanachiwanawin W., Siripanyaphinyo U., Pattanapanyasat K., Wilairat P., Issaragrisil S. Immunophenotypic discrepancies between granulocytic and erythroid lineages in peripheral blood of patients with paroxysmal nocturnal haemoglobinuria. Even so, PHN put people at risk for serious and sometimes life-threatening illnesses. Asystematic analysis of bone marrow cells by flow cytometry defines a specific phenotypic profile beyond GPI deficiency in paroxysmal nocturnal hemoglobinuria. In Paroxysmal Nocturnal Hemoglobinuria (PNH), a clonal marrow stem cell population gives rise to circulating mature hematopoietic cells lacking the expression of a variety of different cell surface proteins whose common feature is their linkage to the cell membrane via a glycosyl-phosphatidyl-inositol (GPI) linkage, a linkage that is deficient in the PNH clone. Regular monitoring of PNH patients, including those receiving eculizumab. HHS Vulnerability Disclosure, Help The molecular basis of paroxysmal nocturnal hemoglobinuria. Testing includes but is not limited to immunodeficiency panels, leukemia/lymphoma panels, quantitative PNH assays, and DNA analysis. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder that happens when part of your immune system attacks and damages red blood cells and platelets. Ed. People who have PNH are six times more likely to develop chronic kidney disease. The FLAER assay is used only in WBC studies, as RBC express glycophorin A, a non-GPI-AP that can weekly bind aerolysin [103]. Ravetch J.V., Perussia B. The flaw launches a cascade of events that create serious and potentially life-threatening medical issues. The flow cytometry results are then used to diagnose or monitor a specific health condition. Patients with PNH may present with a variety of signs and symptoms, including intravascular hemolysis, hemolytic anemia, neutropenia, thrombocytopenia, renal failure, arterial and pulmonary hypertension, smooth muscle dystonia, infections and thrombosis, among others. (iii) Neutrophils should always be studied; RBC testing in recommended at least in cases with a detectable PNH-clone in WBC; monocyte testing may not be suitable for high sensitivity analysis because of the difficulty in collecting sufficient events; routine analysis for RBC only is not recommended; lymphocyte and platelet testing are not adequate for diagnosis. Johnson S.T. urinary stones, infections) that may contribute to the deterioration of renal function. Sutherland D.R., Keeney M., Illingworth A. Richards S.J., Rawstron A.C., Hillmen P. Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. There are a number of common uses for flow cytometry: Flow cytometry analysis is used to count the number of CD4 T-cells in the blood of someone with HIV. Some samples can be taken at your healthcare provider's office but others will need to be done at an outpatient surgical center. The main reason we use anti-CD10 instead of anti-CD15 (currently recommended) for neutrophil selection is because, as mentioned before, anti-CD15 mAbs causes neutrophil aggregation, requiring a careful antibody titration for each batch of mAb used [27,31]. It is important to contact your healthcare provider if you experience any of the following after a bone marrow test or other biopsy: If you have mild to moderate pain after one of these procedures, it should generally be manageable with over-the-counter pain medications. hepatic veins/Budd-Chiari syndrome, other intra-abdominal veins (e.g. Most patients have only type I and type III cells; some patients have type I, type II and type III cells (the second most common phenotype); and a few patients have only type I and type II cells [83]. 2017 Dec;33(4):453-462. doi: 10.1007/s12288-017-0868-y. Even with treatment, people who have PNH need to take steps to prevent blood clots. Epub 2021 Feb 22. Although these ancillary tests were used for a long time for the diagnosis of PNH, they are in disuse, mainly because they have a relatively low sensitivity, as compared to FCM [60]. chronic hemolytic anemia with paroxysmal nocturnal hemoglobinuria: certain immunological aspects of the hemolytic mechanism with special reference to serum complement. Limitations This test was developed, and its performance characteristics determined, by LabCorp. Thrombosis with unusual features and/or occurring at unusual sites e.g. Paroxysmal Nocturnal Hemoglobinuria (PNH) - Cancer Therapy Advisor FSC and SSC, represented as FSC and SSC integral, were captured on a logarithmic scale; for fluorescence parameters, a logarithmic amplification was also used. Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare blood disorder characterized by chronic intravascular hemolysis, thromboses in unusual sites and cytopenias related to bone marrow failure. Cytom. Krawitz P.M., Hchsmann B., Murakami Y., Teubner B., Krger U., Klopocki E., Neitzel H., Hoellein A., Schneider C., Parkhomchuk D., Hecht J., Robinson P.N., Mundlos S., Kinoshita T., Schrezenmeier H. Acase of paroxysmal nocturnal hemoglobinuria caused by a germline mutation and a somatic mutation in PIGT. In addition, false positive results are seen in the Hams test in some hematological disorders such as megaloblastic anemia, spherocytosis and HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test, or congenital dyserythropoietic anemia type II). A close association between PNH and the BM failure syndromes, such as aplastic anemia (AA), and the fact that auto-reactive GPI-specific T cells have been identified in PNH and in AA, would suggest an immune-mediated mechanism by which GPI-AP deficient cells are resistant to the attack of cytotoxic T lymphocytes, enabling them to emerge [[9], [10], [11]]. and CD59 is the GPI-linked antigen. For a bone marrow aspiration and biopsy, you may see a healthcare team that includes other specialists. Gargiulo L., Papaioannou M., Sica M., Talini G., Chaidos A., Richichi B., Nikolaev A.V., Nativi C., Layton M., de la Fuente J., Roberts I., Luzzatto L., Notaro R., Karadimitris A. Glycosylphosphatidylinositol-specific, CD1d-restricted T cells in paroxysmal nocturnal hemoglobinuria. Involved in cell adhesion and CTL-target cell conjugate formation. Diagnosing PNH with FLAER and multiparameter flow cytometry The author declares that there is no conflict of interest regarding the publication of this paper.