Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer

2008 ◽  
Vol 74B (2) ◽  
pp. 91-103 ◽  
Author(s):  
Sven Björnsson ◽  
Saga Wahlström ◽  
Eva Norström ◽  
Ingela Bernevi ◽  
Ulla O'Neill ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometer ◽  
Color Flow ◽  
Single Tube

Improved Recognition of Hematogones from B Lymphoblastic Leukemia By 5-Color Flow Cytometric Analysis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1732-1732
Author(s):  
Amanda Lo ◽  
Matthew Miller ◽  
Melissa Cervania ◽  
Quanlin Li ◽  
Mark David Ewalt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Antigen Expression ◽  
Internal Controls ◽  
Mean Fluorescent Intensity ◽  
Color Flow ◽  
Fluorescent Intensity ◽  
Single Tube ◽  
Expression Levels ◽  
Antigenic Expression

Abstract Background: Hematogones (HG) are benign precursor B-cells that are seen in increased numbers in the bone marrow during childhood, following chemotherapy or bone marrow transplant, in certain immune deficiencies, and in autoimmune disorders. Flow cytometry typically shows expression of TdT, CD10, CD19, variable CD20, dim CD22, CD34 (early HG), and dim CD45. As this phenotype is also seen in patients with B lymphoblastic leukemia (B-ALL), it causes a significant problem in distinguishing leukemic blasts from HG, particularly in a regenerating marrow. Furthermore, hematogones are usually more numerous at baseline in younger patient populations, the same age group with a relatively higher incidence of B-ALL. Despite guidance by earlier studies using the above markers for differentiating HG from B-ALL, these markers are not always sufficient and may hinder correct interpretation. Previous studies demonstrate CD58 is commonly expressed in B-ALL but not in hematogones; however, CD58 as a single marker is somewhat limited when expressed at lower levels. In order to improve diagnostic accuracy, we generated a five color antibody panel including CD10, CD19, CD45, CD38, and CD58 to assess the utility of a single tube panel in distinguishing B-ALL from HG. Design: A total of 35 cases with immature B-cell populations, 16 B-ALL (diagnostic and residual/relapsed cases) and 19 HG, were analyzed by 5-color flow cytometry. 32/35 cases were bone marrow aspirates and 3/35 cases were peripheral blood. A single tube containing CD10 FITC/CD58 PE/CD19 ECD/CD38 PC5/CD45 PC7 was analyzed together with the standard acute leukemia panels. To eliminate technical and fluorochrome variability in expression level analysis, relative antigenic expression was determined through comparison with appropriate internal controls. Antigen expression, as measured by the geometric mean fluorescent intensity (MFI), was then compared between B-ALL and HG using the Mann-Whitney U-test to assess for significant difference. Results were correlated with the morphologic, immunohistochemical, cytogenetic, and molecular findings for precise diagnostic classification as HG or leukemia. Results: HG demonstrated significantly brighter expression of CD38 (p<0.01) than that seen in B-ALL. In contrast, B-ALL expressed significantly brighter CD58 (p <0.01) than HG, which showed dim to no expression of the antigen. HG also showed significantly bright expression of CD10 relative to internal control granulocytes; however, this level of expression was similar to that seen in B-ALL. Median antigen expression. Hematogones show bright CD38, but dim to no CD58. Conversely, B-ALL expresses very dim CD38 and variable CD58. CD10 expression, though, demonstrates overlap between the two populations. B-ALL = B lymphoblastic leukemia, MFI = mean fluorescent intensity Comparative antigenic expression levels in hematogones and B-ALL. Select representative histograms showing HG and B-ALL blasts for the antigens CD38, CD58, and CD10 were selected from various patients studied based on those with the closest relative MFI to the overall median detected for that population in the study. The far right column shows the distribution in MFI of relative antigen expression exhibited the populations studied. HG show significantly brighter CD38 expression than B-ALL does (p<0.01). While B-ALL generally expresses brighter CD58 relative to internal controls, expression levels are variable. HG, though, show significantly dimmer CD58 to essentially no CD58 expression, compared to B-ALL (p<0.01). Similar to CD38, HG demonstrate significantly bright CD10, while B-ALL shows overall bright CD10 but variable expression levels amongst studied cases. These expression levels for CD10 overlap between HG and B-ALL and show no real statistical difference. Conclusions: The combination of CD38 and CD58 in a single tube increases the diagnostic accuracy in differentiation of HG from B-ALL. Without utilizing both antigens together, certain cases would have been difficult to interpret. Based on this analysis, we recommend that these markers be utilized in the routine evaluation for acute lymphoblastic leukemia. This is especially critical in post treatment cases in order to avoid misdiagnosis. Furthermore, the use this single tube panel would cut costs while at the same time improve patient care. Table Table. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Improved Concordance of Minimal Residual Disease Measurements By Quantitative PCR and 10-Color Flow Cytometry in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2614-2614
Author(s):  
Mary Sartor ◽  
Draga Barbaric ◽  
Tamara Law ◽  
DR Anuruddhika Dissanayake ◽  
Nicola C Venn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Residual Disease ◽  
Rank Correlation ◽  
Color Flow ◽  
Single Tube ◽  
Time Points ◽  
Colour Flow ◽  
Risk Patients ◽  
Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) after induction and consolidation therapy is highly predictive of outcome for childhood acute lymphoblastic leukaemia (ALL) and is used to identify high risk patients in most current ALL clinical trials. Two methods broadly applicable for MRD analysis in ALL cases are real-time quantitative PCR based detection of unique immunoglobulin and T-cell receptor gene rearrangements (Ig/TCR PCR-MRD) and the multi-parameter flow cytometry based quantitation of Leukemia Associated Immunophenotypes (LAIP Flow-MRD). We compared the two techniques using samples from patients referred for PCR-MRD analysis initially using 4-tube 4-colour flow and more recently 1-tube 10-color flow. Methods: Newly diagnosed consented ALL patients enrolled on ANZCHOG ALL8 (2002-2011) or AIEOP-BFM ALL 2009 (2012-2014) had duplicate bone marrow aspirates, collected at diagnosis, day 15, day 33 and day 79, and analysed by PCR-MRD and Flow-MRD techniques. PCR-MRD analysis utilized clone specific primers and generic probes for Ig/TCR rearrangements according to EuroMRD guidelines. Flow-MRD which detects levels of aberrant combinations of cell-surface proteins using fluorescently labelled antibodies was performed until 2009 with 4-tube 4-colour flow before we adopted a 1-tube approach (9-colour for BCP-ALL and 10-colour T-ALL) based on the AIEOP-BFM harmonised protocol for 2012-2014. Results: Our early comparison showed a relatively poor correlation of 4-colour Flow-MRD results with PCR-MRD (Spearman rank correlation coefficient rho = 0.516, n=267) for patients enrolled at a single centre on ANZCHOG ALL8 in 2002-2009. Only the PCR-MRD results were used for the MRD risk-adapted stratification for patients on this trial. Flow-MRD for subsequent patients on this trial (2010-11) was improved by using more antibodies and adopting a single tube approach. In our current trial, day 15 Flow-MRD results are used for the early identification of low risk patients for a randomized treatment reduction. In bone marrow samples from patients enrolled on this trial, the correlation of the PCR-MRD and Flow-MRD methods is high when considered for all time points (rho = 0.803 n=418; Figure 1). In the same set of patient samples, the concordance between 2 different PCR markers based on different rearrangements was even better (rho = 0.929, n=390). A comparison of time points found that the best correlation between the two methods was observed at day 15 when MRD is often higher and the bone marrow is not regenerating (Table 1). Both PCR and 10-colour flow enabled MRD to be performed for 94% of ALL patients, and only one patient did not have a sensitive MRD assay. Conclusion: The adoption of new approaches to measurement of Flow-MRD, using a single tube and 10-colors, for ALL patients has greatly improved the concordance of Flow-MRD and PCR-MRD results. It is not surprising given the different nature of the techniques that the correlation of results produced by two different markers for PCR-MRD is higher than that with Flow. However we conclude that these two methods can now be used interchangeably at day 15 in BFM-style protocols for ALL patients. The concordance at later time points is weaker and warrants investigation in the whole trial cohort to enable effects of ALL subtype and patient outcomes to be evaluated. Table 1. Concordance of MRD levels at different time points in the same set of patients (Spearman's Rank correlation coefficient rho). MRD by PCR first Ig/TCR marker versus MRD by 10-colour flow MRD by first Ig/TCR PCR marker versus second Ig/TCR marker All timepoints 0.803 (n=418)** 0.921 (n=390)** Day 15 0.795 (n=155)** 0.950 (n=129)** Day 33 0.417 (n=137) 0.826 (n=132)** Day 79 0.383 (n=126) 0.842 (n=129)** ** Correlation is significant at the 0.01 level (2 tailed) Support: NHMRC Australia APP1057746 and Tour De Cure Foundation Figure 1. Comparison of MRD levels measured by 1-tube 10-color Flow MRD versus PCR MRD (left) or by two different PCR Ig/TCR MRD markers (right) in the 418 and 390 paired measurements in the same set of patients. Figure 1. Comparison of MRD levels measured by 1-tube 10-color Flow MRD versus PCR MRD (left) or by two different PCR Ig/TCR MRD markers (right) in the 418 and 390 paired measurements in the same set of patients. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

A Single Tube Antibody Panel for Differential Count by Flow Cytometry.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4938-4938
Author(s):  
Sven Bjornsson ◽  
Saga Wahlstrom ◽  
Ingela Bernevi ◽  
Per Simonsson
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Flow Cytometer ◽  
Multiparameter Flow Cytometry ◽  
Cell Phenotype ◽  
Differential Count ◽  
Single Tube ◽  
Microscopic Evaluation ◽  
Cell Counter

Abstract Introduction Several techniques are used for classification of leukocytes, i.e. microscopy, cell counters and flow cytometry. A skilled microscopist will classify at least 14 cell classes in bone marrow or blood. We have designed a one tube immunophenotyping panel for leukocyte classification and validated it against microscopy. Multiparameter flow cytometry provide at least seven measurements on each cell passing through the laser beam. Our goal was to use the seven parameters available in our flow cytometer to provide a reliable differential count using only one tube. Materials and Methods The FL-1 to FL-4 channels was used for the antibodies CD36-FITC, CD203-PE, CD138-PE, CD45-ECD, CD16-Pcy5 and CD56-Pcy5. FL-5 was used for the DNA-stain DRAQ5. We used a lyse no wash method to ensure minimal loss of fragile cells and 40000 events were aquired a on a Beckman Coulter FC500 flow cytometer with live gating on DRAQ5-positive cells to aquire only nucleated cells. Sequential gating was used to classify cells according to immunophenotypes given in Table 1. Peripheral blood samples collected in EDTA-tubes were analyzed on the Beckman Coulter LH750 cell counter. Cell counter differentials containing >2% erythroblasts (NRBC) was used to select 79 pathological samples included in the study. An extended microscopic evaluation following the NCCLS-H20A protocol was performed on two different slides using Cellavision DiffMaster™ Octavia digitilized microscopy. Results There was a linear correlation within each cell class between flow cytometry and morphology. The correlation coefficients (R-values) given in table 1 seem reasonable for cellular classification. There was a very good correlation between flow cytometric and morhpological classification of immature red cells (NRBC). There was, however, a low correlation for immature granulocytes and basophilic granulocytes. Immature granulocytes was defined as CD16-negative granulocytes in flow cytometry and exceeded the sum of promyelocytes, myelocytes and metamyelocytes in microscopy in all cases. Basophilic granulocytes was defined as CD203-positive events in flow cytometry, and as granulocytes with basophilic granula in microscopy. Flow cytometry basophil values were either lower or higher then microscopical values in the lower measuring range i.e. <1,5% basophils. Table 1 Conclusions Flow cytometry is often used to detect malignant cells but may as well be used to classify normal cells in blood and bone marrow. The use of antibodies will allow accurate classification even if cellular morphology is pertuberated by disease or drug treatment but the extensive antibody panels commonly used are expensive and labour intensive. The seven parameters available in one single tube in a modern cytometer seem to be enough for reliable leucocyte classification even in difficult pathological samples. The main discrepancy between the methods seems to be in the classification of immature granulocytes and basophils. Table 1 Cell Phenotype R-value (flow/morpholgy) N = 79 1 Monocytes CD36+++/SSC+/CD45++ 0,8686 2 Erythroblasts (NRBC) CD36++/CD45+ 0,9560 3 Neutrophiles, all stages SSC+++/CD45++ 0,9473 4 Neutrophiles, band + segm SSC+++/CD45++ /CD16+++ 0,8445 5 Immature granulocytes (promyelo/myelo/metamyelo) SSC+++/CD45++/CD16− 0,6975 6 Eosinophiles SSC+++/CD45+++/CD16− 0,8532 7 Plasma cells CD138++/CD45− - 8 Blasts CD36−/CD16−/CD203−/CD138−/CD45++/SSC− 0,8033 9 Basophiles CD203+/CD45++/SSC+ 0,6038 10 Lymphocytes CD203−/CD138−/CD45+++/SSC− 0,9741 11 NK-cells CD16+/CD56+/CD45+++/SSC− -

scholarly journals Standardization of 8-color flow cytometry across different flow cytometer instruments: A feasibility study in clinical laboratories in Switzerland

2019 ◽  
Vol 475 ◽  
pp. 112348 ◽  
Author(s):  
Hana Glier ◽  
Ingmar Heijnen ◽  
Mathieu Hauwel ◽  
Jan Dirks ◽  
Stéphane Quarroz ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Feasibility Study ◽  
Flow Cytometer ◽  
Color Flow ◽  
Clinical Laboratories

Follicular Lymphoma in Staging Bone Marrow Specimens: Correlation of Histologic Findings With the Results of Flow Cytometry Immunophenotypic Analysis

2007 ◽  
Vol 131 (2) ◽  
pp. 282-287
Author(s):  
Dan Iancu ◽  
Suyang Hao ◽  
Pei Lin ◽  
S. Keith Anderson ◽  
Jeffrey L. Jorgensen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Bone Marrow Biopsy ◽  
Cell Population ◽  
Color Flow ◽  
Flow Cytometric Immunophenotyping ◽  
Histologic Evidence ◽  
Flow Cytometric ◽  
Biopsy Specimens

Abstract Context.—Bone marrow (BM) examination is part of the staging workup of lymphoma patients. Few studies have compared BM histologic findings with results of flow cytometric immunophenotyping analysis in follicular lymphoma (FL) patients. Objective.—To correlate histologic findings with immunophenotypic data in staging BM biopsy and aspiration specimens of FL patients. Design.—Bone marrow biopsy specimens of untreated FL patients were reviewed. Histologic findings were correlated with 3-color flow cytometric immunophenotyping results on corresponding BM aspirates. Results.—Bone marrow biopsy specimens (with or without aspirates) of 114 patients with histologic evidence of FL in BM were reviewed. There were 76 bilateral and 38 unilateral biopsies performed, resulting in 190 specimens: 187 involved by FL and 3 negative (in patients with a positive contralateral specimen). The extent of BM involvement was &lt;5% in 32 (17.1%), ≥5% and ≤25% in 102 (54.6%), &gt;25% and ≤50% in 27 (14.4%), and &gt;50% in 26 (13.9%) specimens. The pattern of involvement was purely paratrabecular in 81 (43.3%), mixed in 80 (42.8%), and purely nonparatrabecular in 26 (13.9%). Immunophenotyping was only performed unilaterally, on BM aspirates of 92 patients, and was positive for a monoclonal B-cell population in 53 (57.6%) patients. Immunophenotyping was more often negative when biopsy specimens showed FL with a purely paratrabecular pattern. For comparison, we assessed 163 FL patients without histologic evidence of FL in BM also analyzed by flow cytometric immunophenotyping. A monoclonal B-cell population was identified in 5 patients (3%). Conclusions.—Our data suggest that 3-color flow cytometric immunophenotyping adds little information to the evaluation of staging BM specimens of FL patients.

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 Recombinant human interleukin-11 stimulates megakaryocytopoiesis and increases peripheral platelets in normal and splenectomized mice

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 901-908 ◽  
Author(s):  
TY Neben ◽  
J Loebelenz ◽  
L Hayes ◽  
K McCarthy ◽  
J Stoudemire ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Color Flow ◽  
Platelet Counts ◽  
In Vivo Treatment ◽  
Interleukin 11 ◽  
Megakaryocyte Progenitors

Abstract The effects of recombinant human interleukin-11 (rhIL-11) on in vivo mouse megakaryocytopoeisis were examined. Normal C57Bl/6 mice and splenectomized C57Bl/6 mice were treated for 7 days with 150 micrograms/kg rhIL-11 administered subcutaneously. In normal mice, peripheral platelet counts were elevated compared with vehicle-treated controls after 3 days of rhIL-11 treatment and remained elevated until day 10. Splenectomized mice treated with rhIL-11 showed elevated peripheral platelet counts that were similar in magnitude to normal rhIL-11-treated mice. However, on day 10 the platelet counts in rhIL-11- treated, splenectomized mice were no longer elevated. Analysis of bone marrow megakaryocyte ploidy by two-color flow cytometry showed an increase, relative to controls, in the percentage of 32N megakaryocytes in both normal and splenectomized animals treated with rhIL-11. In normal mice, the number of spleen megakaryocyte colony-forming cells (MEG-CFC) were increased twofold to threefold relative to controls after 3 and 7 days of rhIL-11 treatment, whereas the number of bone marrow MEG-CFC were increased only on day 7. The number of MEG-CFC in the bone marrow of rhIL-11-treated, splenectomized mice was increased twofold compared with controls on both days 3 and 7 of the study. These data show that in vivo treatment of normal or splenectomized mice with rhIL-11 increased megakaryocyte progenitors, stimulated endoreplication of bone marrow megakaryocytes, and increased peripheral platelet counts. In addition, results in splenectomized mice showed that splenic hematopoiesis was not essential for the observed increases in peripheral platelets in response to rhIL-11 administration.

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

scholarly journals E-cadherin is functionally involved in the maturation of the erythroid lineage.

1995 ◽  
Vol 131 (1) ◽  
pp. 243-249 ◽  
Author(s):  
S Armeanu ◽  
H J Bühring ◽  
M Reuss-Borst ◽  
C A Müller ◽  
G Klein
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Cell Lineage ◽  
Unexpected Finding ◽  
Color Flow ◽  
Pcr Screening ◽  
Maturation Stages ◽  
Differentiation And Proliferation ◽  
E Cadherin

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E-cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E-cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.

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