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Hematopoietic cell phenotyping using free circulating cellular markers

Inactive Publication Date: 2007-02-22
QUEST DIAGNOSTICS INVESTMENTS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010] A panel of CD markers that can be used to determine a CD phenotype in accordance with the methods of the invention are two more. In a preferred embodiment, if only two CD markers are evaluated, then the two CD markers are not CD20 and CD52. The number of CD markers that can be tested is simply limited by the number of CD markers known for which antibodies or other detection agents are available. In a preferred embodiment, only two CD markers are evaluated. The number of CD markers evaluated in a single bodily fluid may easily include three, four, five, six, seven or even more CD markers.
[0013] One of skill in the art would readily recognize that the measurement of multiple CD markers can be accomplished using various types of assays well-known in the art. In preferred embodiments, the CD marker is detected using a specific binding agent, preferably an antibody. In another embodiment, the assay is an immunoassay such as an enzyme-linked immunosorbent assay (ELISA) or sandwich-type ELISA. These assays are particularly amenable to the detection of multiple antigens. In another embodiment, the assay can be flow cytometry. In the later case, a sandwich-type assay involving capture of an antibody-antigen complex on a bead or microparticle and binding of a labeled second antibody can provide useful assay materials to be evaluated by flow cytometry.

Problems solved by technology

The number of CD markers that can be tested is simply limited by the number of CD markers known for which antibodies or other detection agents are available.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Determination of a CD Phenotype in a Patient Having a Proliferative Disorder

[0049] Antibodies to CD markers, for example, CD3, CD4, CD5, CD7, and CD8, are obtained through commercial sources and are immobilized in distinct wells of a 96-well clear methacrylate microplate by the following process. The antibody to be coated onto the plate is diluted to a concentration of 2-10 μg / ml in a buffer such as phosphate buffered saline (PBS) and added to each well at a volume of about 100 μl per well. The plate is incubated for 18-20 hours at room temperature or 4° C. The unoccupied sites are blocked with a blocking agent (200-300 μl / well) such as bovine serum albumin (BSA).

[0050] A test sample of bodily fluid from a patient with a proliferative disorder is serially diluted and added to each well (50-100 μl / well). The plate is incubated for one or more hours. The wells are washed using PBS with 0.05% Tween 20. A second antibody to the same CD marker which is labeled with horse radish peroxid...

example 2

Identification of a Tumor Type Using the CD Phenotype

[0053] The CD phenotype of a proliferative disorder determined using body fluid as described in Example 1 is used to classify the type of proliferative disorder in the individual by comparison to known cell surface CD marker phenotypes. For example, Table 1 and Table 2 provide cell-surface CD marker phenotypes for exemplary types of leukemias. The particular CD phenotype determined in a body fluid from an individual with a proliferative disorder can be compared to the table to identify the classification of the proliferative disorder. For example, if a phenotype is determined in a body fluid sample from an individual with a proliferative disorder to have increased levels in the fluid of CD3 CD4, CD5, and CD7, it can be concluded using the Table 2 that the individual has adult T cell leukemia.

TABLE 1Cell-surface expression of markers in leukemicB-cell proliferative disorders.DiagnosisAntigenCLLPLLHCLFLMCLLP-ICPCLCD5+− / +w−−++ / −−C...

example 3

Monitoring of Treatment or Disease Progression

[0056] To monitor disease progression in the case of a proliferative disorder, bodily fluid samples from a patient are tested at two points in time (i.e., a first test sample and a second test sample). A sample is obtained prior to treatment (first test sample) and following treatment (second test sample) and evaluated to determine the concentration of CD markers as described in Example 1. An increase in the concentration of CD markers in the second test sample, as compared to the first test sample, indicates progression of the disease whereas a decrease in the concentration of CD markers in the second sample versus the first test sample indicates a regression of the disease.

[0057] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual public...

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Abstract

The present invention provides methods of identifying cluster of differentiation (CD) marker phenotype for hematopoietic cells using multiple soluble CD markers circulating in bodily fluid. In particular aspects, the CD marker phenotype can be used to classify the tumor type of a patient having a proliferative disorder. In other aspects, treatment and disease progression can be monitored by measuring the levels of CD markers in bodily fluids of a patient over time.

Description

FIELD OF THE INVENTION [0001] The present invention relates to the determination of cell surface cluster of differentiation (CD) antigen phenotype of a hematopoietic disorder using CD markers circulating in bodily fluid. BACKGROUND OF THE INVENTION [0002] The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention. [0003] Cluster of differentiation (CD) markers have been established to define human leukocyte differentiation antigens (Bernanrd and Boumsell, 1984) by the comparison of reactivities of monoclonal antibodies directed against these antigens. These antigens are expressed on the cell surface of leukocytes and, therefore, serve as markers of cell lineage and distinguish populations of leukocytes with different functions, e.g., neutrophils and monocytes. [0004] Leukocyte cell-surface antigens are widely used clinically for the identification of leukoc...

Claims

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Application Information

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IPC IPC(8): G01N33/567
CPCG01N33/57426G01N33/57488G01N2333/70596G01N2800/56G01N2333/70517G01N2333/70503G01N2333/7051G01N2333/70514G01N33/6869
Inventor ALBITAR, MAHER
Owner QUEST DIAGNOSTICS INVESTMENTS INC
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