Chapter 3 Cell Preparation for the Identification of Leukocytes

Stewart, Carleton C.; Stewart, Sigrid J.

doi:10.1016/s0091-679x(08)61708-2

Cited by 56 publications

(39 citation statements)

References 7 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Isotype controls are antibodies of the same class (isotype) of immunoglobulin as the specific antibody, but are either myeloma-derived antibodies of unknown specificity, or are raised against an antigen (e.g., T2-mycotoxin or dinitrophenyl) that is presumed not to be present on or in the cells under study (35). The ideal isotype control should ''match'' the specific antibody not only in heavy chain (IgA, IgG, IgD, IgE, or IgM), subclass and light chain (kappa, lambda) class but also in fluorochrome type and number of fluorochrome molecules per immunoglobulin (F/P ratio).…”

Section: Isotype Controlsmentioning

confidence: 99%

Considerations for the control of background fluorescence in clinical flow cytometry

Hulspas¹,

O’Gorman

Wood

et al. 2009

Cytometry Part B Clinical

218

190

View full text Add to dashboard Cite

Section: Isotype Controlsmentioning

confidence: 99%

Considerations for the control of background fluorescence in clinical flow cytometry

Hulspas¹,

O’Gorman

Wood

et al. 2009

Cytometry Part B Clinical

218

190

View full text Add to dashboard Cite

“…29,30 In all, 228 patient samples were analyzed with three-color combinations of FITC, PE, and PerCP or PE/Cy5-conjugated mAb. The remaining 20 patient samples (studied after March 1998) were analyzed with four-color combinations of mAb, the fourth fluorophor being allophycocyanin.…”

Section: Multiparameter Flow Cytometry (Mfc)mentioning

confidence: 99%

HLA-DR antigen-negative acute myeloid leukemia

Wetzler

McElwain

Stewart³

et al. 2003

Leukemia

View full text Add to dashboard Cite

Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical significance of HLA Class II antigen expression by AML cells is not known. Therefore, we sought to characterize cases of AML without detectable HLA-DR expression. Samples from 248 consecutive adult AML patients were immunophenotyped by multiparameter flow cytometry at diagnosis. HLA-DR antigens were not detected on AML cells from 43 patients, including 20 with acute promyelocytic leukemia (APL), and 23 with other subtypes of AML. All APL cases had t(15;17), but there were no characteristic chromosome abnormalities in non-APL cases. No direct expression of other antigens was identified in HLA-DR-negative APL and non-APL cases. Interestingly, cells from three HLA-DR-negative non-APL patients had similar morphology to that of the hypogranular variant of APL. This morphology, however, was not present in any HLA-DR-positive AML cases. Treatment response was similar in the 23 HLA-DR-negative non-APL and the 205 HLA-DR-positive patients. Finally, relapse was infrequently associated with changes in HLA-DR antigen expression, as the HLA-DR antigen was lost at relapse in only 4% of HLA-DR-positive cases, and was gained at relapse in only 17% of HLA-DR-negative cases. We conclude that HLA-DR-negative AML includes approximately equal numbers of APL and non-APL cases, and that the morphology of HLA-DR-negative non-APL cases can mimic the hypogranular variant of APL. The diagnosis of APL cannot be based on morphology and lack of HLA-DR antigen expression; rather, it requires cytogenetic or molecular confirmation.

show abstract

“…To effectively block Fc receptor binding (16), blocking serum should contain sufficient Ig of the correct isotype and subclass. Stewart (16) recommends using the IgG fraction of serum at a concentration of 30 kg IgG per lo6 cells.…”

Section: Discussionmentioning

confidence: 99%

“…There has been little systematic investigation of the variation in flow cytometric procedures that may be required when examining surface antigens on populations of elicited or activated M4 compared with resident M4 or other leukocytes (11,12,16). The present studies describe selective blocking techniques that are necessary to improve the detection of the commonly studied M4 antigens F4/80 and Mac-2 on mouse peritoneal M+ (PM4) elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE-2).…”

mentioning

confidence: 99%

Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages

1994

View full text Add to dashboard Cite

Blocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PM+) by flow cytometry are not ideal for elicited or activated PM+. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac-1 antigens on resident PM+. In contrast, high concentrations (3350% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PM+ elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE-2). However, even low concentrations of goat serum masked the expression of the Mac-2 antigen on TG and MVE-2 PM+. Thus, within a given elicited or activated PM+ population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE-2 PM+. o

show abstract

Chapter 3 Cell Preparation for the Identification of Leukocytes

Cited by 56 publications

References 7 publications

Considerations for the control of background fluorescence in clinical flow cytometry

Considerations for the control of background fluorescence in clinical flow cytometry

HLA-DR antigen-negative acute myeloid leukemia

Optimizing the detection of cell surface antigens on elicited or activated mouse peritoneal macrophages

Contact Info

Product

Resources

About