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Whole blood preparation for cytometric analysis of cell signaling pathways

A cell counting and cell technology, applied in the analysis of materials, biomaterials analysis, individual particle analysis, etc.

Active Publication Date: 2007-08-29
BECKMAN COULTER INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result, pharmacodynamic monitoring had to measure whole blood samples rather than isolated white blood cells

Method used

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  • Whole blood preparation for cytometric analysis of cell signaling pathways
  • Whole blood preparation for cytometric analysis of cell signaling pathways
  • Whole blood preparation for cytometric analysis of cell signaling pathways

Examples

Experimental program
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Effect test

preparation example Construction

[0020] The present invention relates to a preparation method of a biological sample for the measurement of protein epitopes, which makes it possible to detect signal transduction pathways based on the ability to capture intracellular protein epitopes and transient activation states of epitopes. The methods provided by the present invention allow rapid fixation of biological samples containing erythrocytes to ensure that epitopes of signal transduction molecules and other intracellular protein epitopes remain in an activated state. The method of the present invention further allows lysis of red blood cells, thus making it a useful method for cytometric analysis of biological samples including, for example, whole blood, bone marrow aspirate, peritoneal fluid, and other red blood cell-containing sample. The present invention also provides a means to restore or "expose" epitopes on intracellular antigens that are cryptic due to the cross-linked fixative required to fix the sample....

Embodiment 1

[0062] Lysis and fixation of red blood cells and permeabilization of white blood cells

[0063] This example describes and compares the preparation of whole blood samples by hypotonic lysis followed by fixation and post-fixation detergent lysis.

[0064] Briefly, phorbol myristoyl ethyl ester (PMA, Sigma Chemical Corp., St. Louis, MO) was prepared as a 40 M working solution in 100% absolute ethanol and used in whole blood at a final concentration of 400 nM . Triton X-100 and other detergents used in this example were prepared in Surfact-Pak TM Detergent sample kit formats were purchased from Pierce Biotechnology (Rockford, IL). The sample kit contains seven different nonionic detergents (Tween-20, Tween-80, Triton X-100, Triton X-114, Nonidet P-40, Brij-35 and Brij-58, all named 10% aqueous solution) and three powders (nonionic octyl-B-glucoside, and octyl-B-thioglucopyranoside, and amphoteric CHAPS). The powdered detergent was dissolved in PBS (without Ca 2+ and Mg 2+ ...

Embodiment II

[0089] Effects of Fixative and Detergent Concentrations on Leukocyte Light Scattering

[0090] This example demonstrates the effect of concentration and incubation time of both fixative and detergent on the degree of separation and recovery of leukocytes.

[0091] The effect of different fixative concentrations was investigated following previous studies suggesting that high concentrations of cross-linked fixatives help maintain the light-scattering profile and separation of leukocyte populations. Whole blood samples were incubated in increasing concentrations of formaldehyde (from 1% to 10% final concentration) for 10 minutes at room temperature or 37°C, followed immediately by incubation with 1 ml of 0.1% TX-100 (at room temperature). As shown in Figure 6, gradually increasing the concentration of formaldehyde from 2% to 4% (for room temperature incubation) significantly improved the separation of leukocyte populations (left panel). Similarly, treatment of whole blood sam...

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Abstract

This invention is directed to a method for preparation of a biological sample for measurement of protein epitopes that allows for the preservation of intracellular protein epitopes and detection of signal transduction pathways based on the ability to capture transient activation states of the epitopes. The method provided by the invention allows for the rapid fixation of biological samples containing red blood cells, to ensure that epitopes of signal transduction molecules and other intracellular protein epitopes are preserved in the active state. The method of the invention further allows for lysis of red blood cells, thereby making it a useful method for cytometric analysis of biological samples, including, for example, whole blood, bone marrow aspirates, peritoneal fluids, and other red blood cell containing samples. The invention also provides a method to recover or ''unmask'' epitopes on intracellular antigens that have been made inaccessible by the cross linking fixative necessary to fix the sample. Significantly, the methods of the invention allow preservation and analysis of phospho-epitope levels in biological samples taken directly from patients to determine disease-specific characteristics.

Description

technical field [0001] The present invention generally relates to the field of sample preparation for cytometry, and more specifically, the present invention relates to the preparation of biological samples that can be used for signal transduction measurements using flow cytometry and image cytometry. Background technique [0002] Because of its ability to distinguish subpopulations within heterogeneous cell populations, flow cytometry has become an indispensable tool in clinical and basic immunology research. Recently, significant advances in flow cytometry instrumentation and applications have increased the number of parameters that can be analyzed simultaneously to more than thirteen. With the availability of more parameters, researchers have begun to identify more well-defined and biologically interesting subpopulations of lymphocyte samples based on surface epitope staining. [0003] Flow cytometry is routinely used to identify cell populations based on surface phenoty...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53G01N1/00G01N15/10
CPCG01N33/80Y10T436/107497Y10T436/25375Y10T436/108331Y10T436/25125Y10T436/25Y10T436/2525
Inventor 休·乔戴维·赫德利T·文森特·尚克伊帕特里夏·格罗姆
Owner BECKMAN COULTER INC
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