scholarly journals COVID-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of which correlate with patient outcome

2020 ◽  
Author(s):  
Dan Zhang ◽  
Rui Guo ◽  
Lei Lei ◽  
Hongjuan Liu ◽  
Yawen Wang ◽  
...  
Keyword(s):  
Patient Outcome ◽  
Flow Cytometry ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Blood Monocytes ◽  
Forward Scatter ◽  
Inflammatory Monocytes ◽  
Flow Cytometric ◽  
Functional Differences ◽  
Inflammatory Phenotype

AbstractBackgroundExcessive monocyte/macrophage activation with the development of a cytokine storm and subsequent acute lung injury, leading to acute respiratory distress syndrome (ARDS) is a feared consequence of infection with COVID-19. The ability to recognize and potentially intervene early in those patients at greatest risk of developing this complication could be of great clinical utility.MethodsWe performed detailed flow cytometric analysis of peripheral blood samples from 28 COVID-19 patients treated at Xian No.8 Hospital and the First Affiliated Hospital of Xian Jiaotong University in early 2020 in an attempt to identify factors that could help predict severity of disease and patient outcome.FindingsWhile we did not detect significant differences in the number of monocytes between patients with COVID-19 and normal healthy individuals,we did identify significant morphological and functional differences, which are more pronounced in patients requiring prolonged hospitalization and ICU admission. Patients with COVID-19 have larger than normal monocytes, easily identified on forward scatter, side scatter analysis by routine flow cytometry,with the presence of a distinct population of monocytes with high forward scatter (FSC-high). On more detailed analysis, these FSC-high monocytes are CD11b+, CD14+, CD16+, CD68+, CD80+, CD163+, CD206+ and secrete IL-6, IL-10 and TNF-alpha, consistent with an inflammatory phenotype.ConclusionsThe detection and serial monitoring of this subset of inflammatory monocytes using flow cytometry could be of great help in guiding the prognostication and treatment of patients with COVID-19 and merits further evaluation.

Clinical Predictors of Abnormal Peripheral Blood Lymphocytoses Diagnosed by Flow Cytometry: An Algorithmic Approach That Can Be Applied in Routine Clinical Practice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3932-3932
Author(s):  
Jared M. Andrews ◽  
Mitchel T. Holm ◽  
Jerome B. Myers
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Lymphocyte Count ◽  
Flow Cytometric Analysis ◽  
Clinical History ◽  
Lymphocytic Leukemia ◽  
Western World ◽  
Abnormal Phenotype ◽  
Flow Cytometric ◽  
The Mean

Abstract Background Elevated peripheral blood lymphocyte counts in adults can occur in benign reactive conditions as well as malignant disease processes. Chronic lymphocytic leukemia (CLL) is the most common adult hematologic malignancy of the western world affecting the middle aged and elderly. Less commonly B, T, and Natural Killer (NK) cell leukemia / lymphomas may also present with lymphocytosis. Flow cytometry has greatly improved the ability to detect low levels of abnormal lymphocyte populations in peripheral blood. It is, however, a relatively expensive test and clinical guidelines for its appropriate usage are not well defined. Methods We conducted a retrospective review of peripheral blood lymphocytoses that were submitted for flow cytometric analysis at Madigan Army Medical Center, Tacoma, WA from 2002 – 2004. Under laboratory protocol, all patients ≥ 50 years of age with an absolute lymphocyte count (ALC) of > 4 X 109 Cells/L had a peripheral smear evaluated by both a hematology technician and pathologist. Specimens determined to warrant flow cytometric analysis based on review of clinical history, prior lab values, degree of lymphocytosis, and morphology were either recommended for flow cytometry in a comment; or sent directly for analysis with the clinician’s approval. We reviewed complete blood counts (CBCs), previous flow cytometry results, as well as bone marrow and electronic clinical history. All patients with previous diagnoses of lymphoproliferative disorders (LPDs) or ALC < 4 X 109 Cells/L were excluded. Results Approximately 7,300 CBC specimens/month (3,400 from patients ≥ 50 years of age) were performed. Of these, an average of 44 specimens/month had a lymphocytosis of ≥ 4 X 109 Cells/L, from approximately 28 different patients. From this group 71 flow cytometric cases (an average of 2/month) were performed over the 2 year period. 42 cases (59%) had an abnormal phenotype. 27 had a phenotype consistent with CLL, and the other 15 were a mixture of LPDs involving B and T-lymphocytes as well as NK cells. Comparing normal phenotype to abnormal phenotype showed statistically significant differences between the mean age (n-60.4 ±7.5, abn-69.8±8.7), ALC (n-4.9±0.8, abn-9.2±8.1), and relative lymphocyte count (RLC) (n-43.9±7.5%, abn-59.3±8.8%). Conclusion Absolute lymphocyte counts ≥ 4 X 109 Cells/L in adults ≥ 50 years of age represent approximately 1% of the CBCs performed in our laboratory. Review of these cases by a pathologist is logistically feasible due to the low incidence. Our method of reviewing for morphology, clinical history, and past lymphocyte counts with comments to the ordering clinician yielded a high incidence of abnormal phenotype diagnoses when evaluated by flow cytometric analysis (59%). Age, ALC, and relative lymphocyte counts are variables that can be used to develop guidelines for determining the appropriateness of flow cytometric analysis. Patients < 52.4 years of age fall below two standards of deviation from the mean age of the abnormal phenotype group. The standard of deviation for mean ALC is very small (4.9±0.8), which indicates that counts > two standards of deviation above the mean, or 6.5 X 109 Cells/L, would correlate strongly with an abnormal phenotype. The same conclusion could be made with a RLC > 58.9%. In conclusion, patients ≥ 50 years of age with an ALC > 6.5 X 109 Cells/L or a RLC > 58.9% are likely to have a lymphoproliferative disorder and flow cytometric analysis is indicated.

Characteristics of Laboratory Screening Suggest That Paroxysmal Nocturnal Hemoglobinuria (PNH) Is Perceived Primarily as a Bone Marrow Failure Syndrome Rather Than as a Hemolytic Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4107-4107
Author(s):  
Hwee Yong Lim ◽  
Marjorie Farley ◽  
Carl Wittwer ◽  
Charles Parker
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Small Populations ◽  
Hematopoietic Stem ◽  
Flow Cytometric ◽  
Bone Marrow Failure Syndromes

Abstract PNH is a hematopoietic stem cell disorder in which the predominant clinical manifestations are hemolysis, bone marrow failure and thrombophilia. PNH arises as a result of somatic mutation of PIGA, an X-linked gene required for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that anchors some proteins to the cell surface; and consequently, progeny of affected stem cells are deficient in all GPI-anchored proteins (GPI-APs). The hemolysis of PNH is the result of deficiency of CD55 and CD59, GPI-APs that normally inhibit complement activation on the red cell surface, but the relationship between GPI-AP deficiency and the bone marrow failure and thrombophilia of PNH are enigmatic. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and the degree of mosaicism varies greatly among patients. By using fluorescently labeled antibodies, GPI-AP deficient cells (GPI-AP−) can be distinguished form GPI-AP sufficient cells (GPI-AP+) cells by flow cytometric analysis, allowing quantitation of mosaicism. Flow cytometry has been used diagnostically for more than a decade, and technical modifications have improved resolution so that very small populations of GPI-AP− peripheral blood cells can be accurately detected. The purpose of these studies was to generate insights into how PNH is perceived in the community by analyzing the results of a commercially available screening assay using data from a national clinical diagnostic laboratory (ARUP Laboratories, Salt Lake City, UT). The flow cytometric method used in these studies is a modification of the high-resolution two-color assay of Sugimori and colleagues (Blood2006, 107:1308–1314). Clients are given the choice of testing for PNH by analyzing peripheral blood RBCs or PMNs (or both). The acidified serum test (Ham’s test) and the sucrose lysis test (sugar water test) are also available for screening for PNH. For flow cytometric analysis of RBCs, a value of ≥0.005% GPI-AP− cells is considered abnormal, while for PMNs ≥0.003% is abnormal. From January 1, 2008 to June 30, 2008, 1,113 RBC assays and 133 PMN assays were performed. An abnormally large population of GPI-AP− RBCs was identified in 55 cases (5%). The percentage of GPI-AP− RBC ranged from 0.009–69.603% with a median of 1.405%. Twenty-two cases (40%) had >5% GPI-AP− RBCs, while 18 cases (33%) had >10% GPI-AP− RBCs. Of the 133 PMN assays performed, 15 (11%) were abnormal. The range of GPI-AP− PMNs was 0.004–97.727% with a median of 18.327 %. Eight samples (53%) had >10% GPI-AP− PMNs. During the 1-year period from July 1, 2007-June 30, 2008 the acidified serum lysis test (Ham’s test) was performed on 212 samples while the sucrose lysis test was performed on 148 samples. These studies suggest that screening for PNH is common (~43 RBC assays/week compared to 44 assays/week for flow cytometric screening of peripheral blood for lymphoproliferative disorders and leukemia), but the vast majority of samples tested show normal expression of GPI-APs. That so many of the test samples are negative, and that the median for abnormal RBC samples is ~1.5 % GPI-AP− cells, suggest that most of the screening is done because of the association of PNH with bone marrow failure syndromes rather than because of evidence of intravascular hemolysis. These studies underscore the need to understand the pathophysiological basis and clinical implications of small populations of GPI-AP deficient cells in patients with bone marrow failure syndromes. Nonetheless, 18 cases with >10% GPI-AP− RBCs were detected during the 6 months of observation, indicating that the prevalence of classic PNH in the US is substantial. That PNH clone size is best determined by analysis of GPI-AP expression on PMNs does not appear to be widely appreciated in the community as the PMN assays is requested 12% as often as the RBC assay. Flow cytometry has largely, but not completely, replaced Ham’s test and the sucrose lysis test as screening assays for PNH.

scholarly journals Multiparameter Flow Cytometric Analysis of Antibiotic Effects on Membrane Potential, Membrane Permeability, and Bacterial Counts of Staphylococcus aureus andMicrococcus luteus

2000 ◽  
Vol 44 (4) ◽  
pp. 827-834 ◽  
Author(s):  
David J. Novo ◽  
Nancy G. Perlmutter ◽  
Richard H. Hunt ◽  
Howard M. Shapiro
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Membrane Permeability ◽  
Micrococcus Luteus ◽  
Flow Cytometric Analysis ◽  
Bacterial Counts ◽  
Flow Cytometric ◽  
Permeability Parameter ◽  
Particle Counts

ABSTRACT Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus andMicrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC2(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC2(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 μg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 μg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 μg/ml did not change permeability, while a tetracycline concentration of 4 μg/ml permeabilized 50% of the bacteria; 4 μg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.

Immunophenotypic Study of Basophils by Multiparameter Flow Cytometry

2008 ◽  
Vol 132 (5) ◽  
pp. 813-819
Author(s):  
Xiaohong Han ◽  
Jeffrey L. Jorgensen ◽  
Archana Brahmandam ◽  
Ellen Schlette ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Myelogenous Leukemia ◽  
Multiparameter Flow Cytometry ◽  
Color Flow ◽  
Aberrant Expression ◽  
Flow Cytometric ◽  
Hla Dr

Abstract Context.—The immunophenotypic profile of basophils is not yet fully established, and the immunophenotypic changes in chronic myelogenous leukemia are not fully characterized. Objective.—To establish a comprehensive immunophenotypic spectrum of normal basophils and to assess the range of immunophenotypic aberrations of basophils in chronic myelogenous leukemia. Design.—Using 4-color flow cytometry, we compared the immunophenotypic profile of basophils in peripheral blood or bone marrow samples from 20 patients with no evidence of neoplasia to basophils from 15 patients with chronic myelogenous leukemia. Results.—Basophils in control cases were all positive for CD9, CD13, CD22, CD25 (dim), CD33, CD36, CD38 (bright), CD45 (dimmer than lymphocytes and brighter than myeloblasts), and CD123 (bright), and were negative for CD19, CD34, CD64, CD117, and HLA-DR. Basophils in all chronic myelogenous leukemia patients possessed 1 to 5 immunophenotypic aberrancies. The most common aberrancies were underexpression of CD38, followed by aberrant expression of CD64 and underexpression of CD123. CD34 and CD117 were present in cases with basophilic precursors. Myeloblasts showed a distinct immunophenotypic profile, as they typically expressed CD34 and CD117, showed dimmer expression (compared with basophils) of CD38, CD45, and CD123, and lacked expression of CD22. Conclusions.—Flow cytometric immunophenotyping can identify immunophenotypic aberrations of basophils in chronic myelogenous leukemia, and discriminate basophils from myeloblasts.

scholarly journals Flow Cytometric Analysis of T, B, and NK Cells Antigens in Patients with Mycosis Fungoides

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Serkan Yazıcı ◽  
Emel Bülbül Başkan ◽  
Ferah Budak ◽  
Barbaros Oral ◽  
Şaduman Balaban Adim ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycosis Fungoides ◽  
Peripheral Blood ◽  
Prognostic Value ◽  
Flow Cytometric Analysis ◽  
Surface Antigens ◽  
Cell Surface Antigens ◽  
Flow Cytometric ◽  
Hla Dr ◽  
The Mean

We retrospectively analyzed the clinicopathological correlation and prognostic value of cell surface antigens expressed by peripheral blood mononuclear cells in patients with mycosis fungoides (MF). 121 consecutive MF patients were included in this study. All patients had peripheral blood flow cytometry as part of their first visit. TNMB and histopathological staging of the cases were retrospectively performed in accordance with International Society for Cutaneous Lymphomas/European Organization of Research and Treatment of Cancer (ISCL/EORTC) criteria at the time of flow cytometry sampling. To determine prognostic value of cell surface antigens, cases were divided into two groups as stable and progressive disease. 17 flow cytometric analyses of 17 parapsoriasis (PP) and 11 analyses of 11 benign erythrodermic patients were included as control groups. Fluorescent labeled monoclonal antibodies were used to detect cell surface antigens: T cells (CD3+, CD4+, CD8+, TCRαβ+, TCRγδ+, CD7+, CD4+CD7+, CD4+CD7−, and CD71+), B cells (HLA-DR+, CD19+, and HLA-DR+CD19+), NKT cells (CD3+CD16+CD56+), and NK cells (CD3−CD16+CD56+). The mean value of all cell surface antigens was not statistically significant between parapsoriasis and MF groups. Along with an increase in cases of MF stage statistically significant difference was found between the mean values of cell surface antigens. Flow cytometric analysis of peripheral blood cell surface antigens in patients with mycosis fungoides may contribute to predicting disease stage and progression.

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