Evaluation of a method for counting absolute numbers of cells with a flow cytometer

Nicholson, Janet K. A.; Stein, Dara; Mui, Tammy; Mack, Richard; Hubbard, Marjorie; Denny, Thomas N.

doi:10.1128/cdli.4.3.309-313.1997

Cited by 73 publications

(30 citation statements)

References 11 publications

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“…This alone leads to an FCM-depending imprecision of up to 10%. In a limited investigation of repeatability of FCM on eight samples that were analyzed for CD4 1 T-cells in our laboratory and in the laboratory of the St. George Medical Center in Leipzig, the coefficient of variation was 12.5%, consistent with previous reports (20,32,33). It is therefore possible that imprecision and inaccuracy of MNS are in fact lower than that reported here.…”

Section: Discussionsupporting

confidence: 90%

Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

et al. 2007

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The CD4 count is the best surrogate marker for monitoring HIV. The reference method for assessing CD4 counts (flow cytometry, FCM), as expensive technique, is not widely used in non-OECD countries. To make HIV-monitoring available to more patients in these countries, we modified a commercially available density-based negative selection assay (RosetteSep TM ) to make it applicable for low-cost cell enumeration. For evaluation (Step 1), blood taken from 25 HIV patients and 29 healthy donors was assayed with the modified negative selection method (MNS) and compared with FCM. For validation (Step 2), this method was performed in blind quintuplicates on 12 HIVþ blood samples according to FDA guidelines. Association of MNS with the FCM is given by regression models for both steps:Step 1: slope = 1.091, intercept = À46.5.Step 2: slope = 1.074, intercept = À38.3 (Step 2). The imprecision of MNS assessed during Step 2 was 21.2% (intraserial) and 18.8% (interserial). The results suggest that MNS is capable of providing an approximate CD4 count. At a cost of 0.30, it is affordable to patients living in resource-restrained areas. The technique has the potential to deliver an accurate, precise, low-cost test to monitor HIVþ patients. ' 2007 International Society for Analytical Cytology

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Section: Discussionsupporting

confidence: 90%

Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

et al. 2007

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“…The use of Trucount tubes containing a defined number of fluorescent beads has been validated in clinical practice for measurement of cells and platelets in human blood (36–38). Since the use of SYTO‐16 involves no loss of erythrocytes due to sample processing before acquisition in flow cytometry, experiments were carried out to determine whether Trucount tubes could be used to measure the concentration of erythrocytes in blood in our assay conditions.…”

Section: Resultsmentioning

confidence: 99%

“…As there are no changes in sample volume, the measurement of the actual concentrations of erythrocytes in blood was possible. The use of Trucount tubes was strongly advocated because of its demonstrated reliability in clinical practice to measure the concentration of leukocytes (36), red cell vesicles in thalassemic patients (37), and particularly, platelets or residual cells in plasma products (38). However, given the relatively high cost of these tubes, other approaches could be validated for each particular analytical purpose.…”

Section: Discussionmentioning

confidence: 99%

Quantitative measurement of Plasmodium‐infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO‐16 fluorescence

et al. 2008

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Flow cytometry is a powerful tool for measuring parasitemias in murine malaria models used to test new antimalarials. Measurement of the emission of the nonpermeable nucleic acid dye YOYO-1 (at 530 and 585 nm after excitation at 488 nm) allowed the unambiguous detection of low parasitemias (!0.01%) but required prolonged fixation and permeabilization of the sample. Thus, we tested whether this issue could be overcome by use of the cell-permeant dye SYTO-16 with this same bidimensional method. Blood samples from CD1 mice infected with Plasmodium yoelii, Plasmodium vinckei, or Plasmodium chabaudi or from NOD scidb2m-/-engrafted with human erythrocytes and infected with P. falciparum were stained with SYTO-16 in the presence or absence of TER-119 mAb (for engrafted mice) in 96-well plate format and acquired in Trucount TM tubes. Bidimensional analysis with SYTO-16 was quantitatively equivalent to YOYO-1. Moreover, by combining SYTO-16 with the use of TER-119-PE antimouse erythrocyte mAb and Trucount tubes, the measurement of the concentration of P. falciparuminfected erythrocytes over a range of five orders of magnitude was achieved. Bidimensional analysis using SYTO-16 can be used to accurately measure the concentration of Plasmodium spp.-infected erythrocytes in mice without complex sample preparation. MALARIA is caused by the erythrocytic stages of protozoa of the genus Plasmodium, which colonize and destroy host's erythrocytes (1). To counter this disease, murine models of malaria are essential tools for research (2), particularly for drug discovery (3). In addition to the standard rodent experimental systems, different murine models of P. falciparum malaria are currently available (4-6). These are of special interest for drug discovery because, with the exception of human subjects, these are the only experimental systems available that allow the evaluation in vivo of the real human pathogen growing inside human erythrocytes (hE) previously engrafted into immunodeficient mice. Not surprisingly, the peripheral blood of these chimeric mice [humanized mice (HM)] is a complex mixture of murine erythrocytes (mE) and hE, in which the hematological effects of massive transfusions of hE and their elimination from peripheral blood may have important effects. Hence, the specific and quantitative measurement of different erythrocytic subpopulations is crucial in HM models, particularly when these models are used to establish the relationship between the amount of an antimalarial drug in blood and the effect on parasitemia through experimental pharmacokinetic and pharmacodynamic studies (PK/PD). In this kind of

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“…We transplanted mOVA skin grafts onto B6.OT-I Memory recipients that were treated with an immunosuppression regimen prior to sacrifice on POD 7. Absolute quantification of OT-I T cells from the memory recall response in the blood, draining lymph nodes and spleen was performed using flow cytometry (50). Despite a decrease in the absolute number of OT-I T cells in the blood in treated versus untreated recipients, there were no significant differences between the absolute number of OT-I T cells in the dual costimulatory/integrin blockade recipient groups compared to treatment with CoB alone ( Figure 4A).…”

Section: Combined Integrin and Cob Does Not Curtail Recall Accumulatimentioning

confidence: 99%

Integrin Antagonists Prevent Costimulatory Blockade-Resistant Transplant Rejection by CD8+ Memory T Cells

Kitchens

Haridas

Wagener

et al. 2012

American Journal of Transplantation

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The success of belatacept in late-stage clinical trials inaugurates the arrival of a new class of immunosuppressants based on costimulatory blockade, an immunosuppression strategy that disrupts essential signals required for alloreactive T cell activation. Despite having improved renal function, kidney transplant recipients treated with belatacept experienced increased rates of acute rejection. This finding has renewed focus on costimulatory blockade-resistant rejection and specifically the role of alloreactive memory T cells in mediating this resistance. To study mechanisms of costimulatory blockade-resistant rejection and enhance the clinical efficacy of costimulatory blockade, we developed an experimental transplant system that models a donor-specific memory CD8+ T cell response. After confirming that graft-specific memory T cells mediate costimulatory blockade-resistant rejection, we characterized the role of integrins in this rejection. The resistance of memory T cells to costimulatory blockade was abrogated when costimulatory blockade was coupled with either anti-VLA-4 or anti-LFA-1. Mechanistic studies revealed that in the presence of costimulatory blockade, anti-VLA-4 impaired T cell trafficking to the graft but not memory T cell recall effector function, whereas anti-LFA-1 attenuated both trafficking and memory recall effector function. As antagonists against these integrins are clinically approved, these findings may have significant translational potential for future clinical transplant trials.

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Evaluation of a method for counting absolute numbers of cells with a flow cytometer

Cited by 73 publications

References 11 publications

Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Quantitative measurement of Plasmodium‐infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO‐16 fluorescence

Integrin Antagonists Prevent Costimulatory Blockade-Resistant Transplant Rejection by CD8+ Memory T Cells

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Evaluation of a method for counting absolute numbers of cells with a flow cytometer

Cited by 73 publications

References 11 publications

Low‐cost enumeration of CD4+ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Low‐cost enumeration of CD4+ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Quantitative measurement of Plasmodium‐infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO‐16 fluorescence

Integrin Antagonists Prevent Costimulatory Blockade-Resistant Transplant Rejection by CD8+ Memory T Cells

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Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries

Low‐cost enumeration of CD4⁺ T cells using a density‐based negative selection method (RosetteSep™) for the monitoring of HIV‐infected individuals in non‐OECD countries