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Compositions for permeabilising fixed blood cells and uses thereof

A technology for composition and blood cells, which is applied in the preparation of test samples, biological tests, instruments, etc., and can solve problems such as limiting analysis, destroying cell surface markers, and troublesome.

Active Publication Date: 2015-11-18
BECKMAN COULTER INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, alcohols may also alter cell structure and disrupt cell surface markers
Additionally, the use of alcohols limits subsequent analysis and requires at least one centrifugation step to separate alcohols from treated cells
[0010] Whenever a sample containing red blood cells, such as a peripheral blood sample, is used for analysis, the lysis of these red blood cells is an important but cumbersome step

Method used

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  • Compositions for permeabilising fixed blood cells and uses thereof
  • Compositions for permeabilising fixed blood cells and uses thereof
  • Compositions for permeabilising fixed blood cells and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0168] Exemplary Preparation of Compositions According to the Invention

[0169] The following compositions provide examples of suitable reagents within the scope of the present invention

[0170] A fixative (reagent A) for fixing a biological sample containing leukocytes, preferably a whole blood sample, comprising the following substances in its aqueous solution:

[0171] Sodium chloride 150mM

[0172] Formaldehyde (without methanol) 10% (w / v)

[0173] Reagent A was pH neutral and was filtered using a nylon filter with a pore size of 0.22 μm.

[0174] A cell treatment composition (reagent B) for treating a biological sample containing leukocytes, preferably a whole blood sample, comprising in its aqueous solution:

[0175]

[0176] Reagent B was prepared by dissolving sodium lauryl sulfate in water. Mix all ingredients except sodium perchlorate to adjust pH to 4.4. Sodium perchlorate was then added and any precipitate produced was dissolved by mixing well. NaOH wa...

Embodiment 2

[0187] Labeling of cell surface antigens

[0188] To a volume of 100 µl of whole blood treated with 0.7 mM ethylenediaminetetraacetic acid (EDTA) as an anticoagulant, 65 µl of a fixative (reagent A in Example 1) was added. The mixture was vortexed and incubated at room temperature (18-25°C) for 10 minutes. After 10 minutes, 1 ml of the cell treatment composition (reagent B in Example 1) was added. The mixture was vortexed immediately and allowed to incubate at 37°C for 5 minutes.

[0189] The pH of this anticoagulated blood is about 7.2. After 10 minutes of fixation, the pH of blood is about 6.1 due to the destruction of amino groups. After adding the cell treatment composition and incubating for 5 minutes, the pH of the mixture was about 5.6.

[0190] The obtained mixture was then centrifuged at about 300 g to remove the liquid phase. The pellet was taken out and placed in 100 [mu]l culture medium (Reagent C in Example 1) and dissolved by vortexing. The pH of the obta...

Embodiment 3

[0226] To evaluate the effect of anionic surfactant concentration, various cell treatment compositions as shown in Example 1 were prepared using different SDS concentrations. Table 3 contains data relating to experiments demonstrating the effect of SDS concentration in cell treatment compositions on the exposure of phospho-epitopes, such as p-Stat5. In this setting, optimal exposure and staining of p-Stat5 was obtained using a cell treatment solution with a concentration of 1% w / v (34.7 mM).

[0227] table 3

[0228]

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Abstract

The present invention relates to a cell treatment composition for the permeabilization of fixed blood cells, to the use of said composition, to a method for the treatment of a biological sample comprising fixation of said sample and subsequently contacting said biological sample with said cell treatment composition. The invention further relates and to a kit comprising said cell treatment composition.

Description

[0001] Cross References to Related Applications [0002] This application was filed as a PCT International Patent Application on December 23, 2013, and claims priority to US Patent Application No. 61 / 747,044, filed December 28, 2012, the entire disclosure of which is incorporated herein by reference. technical field [0003] The present invention relates to a cell treatment composition for permeabilization of fixed blood cells, the use of said cell treatment composition and a method for the treatment of a biological sample comprising substances for subsequent intracellular and / or Analysis of extracellular epitopes such as phospho-epitopes on fixed leukocytes. The present invention further relates to a kit comprising said cell treatment composition. Background technique [0004] Recent developments in the field of cellular (eg, blood cell) analysis have been able to provide valuable tools for detailed analysis of cellular structures within and on the cell surface. To detect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30G01N33/53G01N33/569
CPCG01N1/30
Inventor 安德烈亚斯·凡·阿格索文
Owner BECKMAN COULTER INC
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