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Stabilization of cells and biological specimens for analysis

A technique of biology and specimens, applied in the determination/testing of microorganisms, biochemical equipment and methods, and analytical materials, etc.

Inactive Publication Date: 2005-01-26
IMMUNIVEST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One could not speculate that tumor cells circulating in the blood would be as stable as normal blood cells

Method used

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  • Stabilization of cells and biological specimens for analysis
  • Stabilization of cells and biological specimens for analysis
  • Stabilization of cells and biological specimens for analysis

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Stabilization of tumor cells circulating in the blood

[0052] Cyto-Chex TM , StabilCyte TM and TRANSfix TM is an example of three stabilizers that are commercially available and have demonstrated utility in the long-term stabilization of blood cells in blood samples. The stabilizers were optimized to preserve cell size (mainly by reducing shrinkage) and to protect antigens on the cell surface, as determined primarily by flow cytometry techniques. The intended use generally involves direct analysis and does not require extensive manipulation of the sample or enrichment of specific cell populations. In contrast, circulating tumor cells or other rare target cells that are isolated and tested in the present invention include and are determined to be pathologically abnormal or rare cells that occur at very low frequency and therefore require significant enrichment prior to detection.

[0053] CTCs can usually be detected in blood, preferably after storage at room temper...

Embodiment 2

[0064] Preserve sample quality for analysis

[0065] CTCs are present in blood at low frequency and require large sample volumes and efficient enrichment methods for detection. Enrichment methods for CTCs include several washing steps, including magnetic separation methods, which can damage cells and generate debris and clumps due to leakage of DNA from cells. However, it was unexpectedly, and rather startlingly, discovered that gentle upright cylinder mixing of blood tubes on a nutator (the way most hematology laboratories typically keep blood cells in suspension) results in a large number of cells Formation of debris that may interfere with CTC detection and enumeration. In addition, it has been found that incomplete filling of blood draw or analysis tubes further exacerbates this mixed injury, however, no damage occurs in quiescent tubes. Such as image 3 As shown, any mixing of patient samples during transport or other mechanical stress has been shown to unexpectedly in...

Embodiment 3

[0068] Cyto-Chex TM Effect of Stabilizers on Sample Quality of Normal Specimens Mixed for 2-3 Hours

[0069] DAPI staining of the nucleus can usually be detected in permeabilized live or dead cells if the nucleus is intact. DNA staining as stainable aggregates on the outside of the cells can also be detected in cell fragments or cell debris if the DNA leaks outside the cells. Therefore, staining with DAPI, and examining under a fluorescence microscope, can easily check the sample quality.

[0070] Pipette three tubes of blood from normal donors into 10 ml EDTA anticoagulated tubes. Without removing the cap, by using CellStabilize TM The injection device dispenses Cyto-Chex as follows TM Stabilizers are added to a blood test tube. The blood tube is placed in a graduated calibration device and the blood volume can be estimated so that the appropriate amount of stabilizer can be added to the desired final concentration. One needle (27G, 1 / 2 inch) was inserted into the cap o...

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PUM

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Abstract

Compositions and methods for stabilizing rare cells in blood specimens, preserving the quality of blood specimens, and also serving as cell fixatives are disclosed which minimize losses of target cells (for example, circulating tumor cells) and formation of debris and aggregates from target cells, non-target cells and plasma components, thereby allowing more accurate analysis and classification of circulating tumor cells (CTC) and, ultimately, of tumor burdens in cancer patients. Stabilization of specimens is particularly desirable n protocols requiring rare cells enrichment from blood specimens drawn from cancer patients. Exposure of such specimens to potentially stressful conditions encountered, for example, in normal processing, mixing, shaking, delays due to transporting the blood, has been observed to not only diminish the number of CTC but also to generate debris and aggregates in the blood specimens that were found to interfere with accurate enumeration of target cells, if present. Stabilizers are necessary to discriminate between in vivo CTC disintegration and in vitro sample degredation.

Description

[0001] priority information [0002] Pursuant to 35 USC §119(e), this application claims U.S. Provisional Application No. 60 / 314151, filed August 23, 2001, and U.S. Provisional Application No. 60 / 369628, filed April 3, 2002 priority. Both applications are incorporated by reference herein. field of invention [0003] This application relates generally to the field of cell and blood stabilization, and more specifically to the stabilization of rare cells in blood specimens, most particularly to the stabilization of circulating tumor cells (CTCs) in whole blood for subsequent enrichment and analysis. Background of the invention [0004] Tumor cells have been detected in blood as early as 1869, and there is evidence that primary cancers shed tumor cells into the circulation at an early onset stage before clinical symptoms appear. Upon tumor vascularization, tumor cells shed into the circulation can attach and colonize distant sites to form metastases. Said circulating tumor ce...

Claims

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Application Information

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IPC IPC(8): G01N33/48A01N1/00A01N1/02C12Q1/02G01N33/49G01N33/543G01N33/569G01N33/574
CPCA01N1/00G01N33/574G01N33/54333A01N1/0226A01N1/02G01N33/52G01N33/549
Inventor G·C·劳M·赫尔曼H·鲁特纳L·特尔斯塔彭
Owner IMMUNIVEST
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