US5075556A
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US
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Acridine orange derivatives and their use in the quantitation of reticulocytes in whole blood
Assignee: Bayer Corp
•
Filing: 1989-12-01
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Granted: 1991-12-24
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Expires: 2009-12-01
Abstract
Abstract Quaternized derivatives of acridine orange and reagents incorporating such derivatives and their use in quantitatively determining reticulocyte levels in whole blood specimen by fluorescence flow cytometry techniques are disclosed. The quaternized derivatives of acridine orange are of the general formula: ##STR1## wherein Y is bromide (Br - ) or iodide (I - ), and X may be R 1 and/or R 2 substituted benzyl group ##STR2## R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, or X may be hydroxyl ethylene.
Inventors
- 👤 Sophie Fan
- 👤 Gena Fischer
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 00:04
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/80; C; C09; C09B; C09B15/00; Y; Y10; Y10T; Y10T436/00; Y10T436/14; Y10T436/145555
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
61 words
A quaternized derivative of acridine orange of the formula: ##STR10## wherein Y is bromide (Br - ) or iodide (I - ), and wherein X is the R 1 and/or R 2 substituted benzyl group ##STR11## wherein R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, except R 1 and R 2 cannot both by hydrogen.
Claim 2
Dependent Claim
61 words
A quaternized derivative of acridine orange of the formula: ##STR10## wherein Y is bromide (Br - ) or iodide (I - ), and wherein X is the R 1 and/or R 2 substituted benzyl group ##STR11## wherein R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, except R 1 and R 2 cannot both by hydrogen.
Claim 3
Dependent Claim
87 words
A reagent for staining reticulocytes in a whole blood sample for quantitation which reagent is characterized in that it includes an aqueous solution of: a) a quaternized derivative of acridine orange of the formula: ##STR12## wherein Y is bromide (Br - ) or iodide (I - ), and X may be R 1 and/or R 2 substituted benzyl group ##STR13## in which R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, or X may be hydroxyl ethylene, and b) a buffer system.
Claim 4
Dependent Claim
87 words
A reagent for staining reticulocytes in a whole blood sample for quantitation which reagent is characterized in that it includes an aqueous solution of: a) a quaternized derivative of acridine orange of the formula: ##STR12## wherein Y is bromide (Br - ) or iodide (I - ), and X may be R 1 and/or R 2 substituted benzyl group ##STR13## in which R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, or X may be hydroxyl ethylene, and b) a buffer system.
Claim 5
Dependent Claim
15 words
The reagent of claim 2 wherein the buffer system maintains the pH at about 7.0.
Claim 6
Dependent Claim
14 words
The reagent of claim 2 wherein the buffer system comprises paraformaldehyde and potassium oxalate.
Claim 7
Dependent Claim
30 words
The reagent of claim 4 wherein said paraformaldehyde is present at a concentration of about 1.25 g/L and said potassium oxalate is present at a concentration of about 9 g/L.
Claim 8
Dependent Claim
26 words
The reagent of claim 2 wherein said quaternized derivative of acridine orange is present at a concentration of from about 2 μM to about 20 μM.
Claim 9
Dependent Claim
21 words
The reagent of claim 6 wherein said quaternized derivative of acridine orange is present at a concentration of about 7 μM.
Claim 10
Independent Claim
154 words
A method for quantitating reticulocytes in a whole blood sample by flow cytometry which comprises the steps of: (a) mixing a sample of blood to be tested with a reagent comprising an aqueous solution of the quaternized derivative of acridine orange of the formula: ##STR14## wherein Y is bromide (Br - ) or iodide (I - ) , and X is the R 1 and/or R 2 substituted benzyl group ##STR15## in which R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, or X is hydroxyl ethylene, to form a suspension; (b) allowing the suspension to react for a sufficient time so that the derivative is effectively taken up by the reticulocytes; (c) exposing the suspension to radiation from a blue laser light source; (d) measuring the intensity of orange fluorescence from the suspension; and (e) determining the amount or percentage of reticulocytes in the sample from said measurement.
Claim 11
Independent Claim
154 words
A method for quantitating reticulocytes in a whole blood sample by flow cytometry which comprises the steps of: (a) mixing a sample of blood to be tested with a reagent comprising an aqueous solution of the quaternized derivative of acridine orange of the formula: ##STR14## wherein Y is bromide (Br - ) or iodide (I - ) , and X is the R 1 and/or R 2 substituted benzyl group ##STR15## in which R 1 is hydrogen or fluorine, and R 2 is fluorine, trifluoromethyl or hydrogen, or X is hydroxyl ethylene, to form a suspension; (b) allowing the suspension to react for a sufficient time so that the derivative is effectively taken up by the reticulocytes; (c) exposing the suspension to radiation from a blue laser light source; (d) measuring the intensity of orange fluorescence from the suspension; and (e) determining the amount or percentage of reticulocytes in the sample from said measurement.
Claim 12
Dependent Claim
16 words
The method of claim 8 wherein the reaction time in step (b) is about three minutes.
Claim 13
Dependent Claim
12 words
The method of claim 8 wherein the reagent includes a buffer system.
Claim 14
Dependent Claim
15 words
The method of claim 10 wherein the buffer system maintains the pH at about 7.0.
Claim 15
Dependent Claim
14 words
The method of claim 10 wherein the buffer system comprises paraformaldehyde and potassium oxalate.
Claim 16
Dependent Claim
30 words
The method of claim 12 wherein said paraformaldehyde is present at a concentration of about 1.25 g/L and said potassium oxalate is present at a concentration of about 9 g/L.
Claim 17
Dependent Claim
25 words
The method claim 12 wherein said quaternized derivative of acridine orange is present at a concentration of from about 2 μM to about 20 μM.
Claim 18
Dependent Claim
21 words
The method of claim 14 wherein said quaternized derivative of acridine orange is present at a concentration of about 7 μM.
References Cited (Backward Citations)
US4336029A
Cited
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