US4575490A
Expired / Lapsed
US
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One step method for sphering and fixing whole blood erythrocytes
Assignee: Bayer Corp
•
Filing: 1984-02-29
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Granted: 1986-03-11
•
Expires: 2004-02-29
Abstract
Abstract A rapid one step, one reagent method is disclosed for the isovolumetric sphering and fixing of whole blood erythrocytes.
Inventors
- 👤 Leonard Ornstein
- 👤 Young R. Kim
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-01 21:49
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; Y; Y10; Y10T; Y10T436/00; Y10T436/10; Y10T436/101666; Y10T436/107497
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
138 words
A method for treating mammalian red blood cells in a whole blood sample which can be subsequently effectively measured electrooptically for determination of red blood cell volumes which comprises treating in a single step an anticoagulated whole blood sample with an isotonic reagent solution containing both a sphering agent and a fixing agent at a temperature in the range of 4° C. to 37° C. over a reaction period of up to 180 seconds employing a whole blood dilution factor of from 1:300 to 1:4,000, wherein said sphering agent and said fixing agent are present in the reagent at concentrations and at a ratio which provides isovolumetric sphering of the erythrocytes in the sample independent of the concentration of said erythrocytes in the whole blood sample and without inducing lysis of said erythrocytes in the resulting diluted sample.
Claim 2
Independent Claim
138 words
A method for treating mammalian red blood cells in a whole blood sample which can be subsequently effectively measured electrooptically for determination of red blood cell volumes which comprises treating in a single step an anticoagulated whole blood sample with an isotonic reagent solution containing both a sphering agent and a fixing agent at a temperature in the range of 4° C. to 37° C. over a reaction period of up to 180 seconds employing a whole blood dilution factor of from 1:300 to 1:4,000, wherein said sphering agent and said fixing agent are present in the reagent at concentrations and at a ratio which provides isovolumetric sphering of the erythrocytes in the sample independent of the concentration of said erythrocytes in the whole blood sample and without inducing lysis of said erythrocytes in the resulting diluted sample.
Claim 3
Dependent Claim
26 words
The method of claim 1 wherein the concentration of said sphering agent in the reagent is from about 0.5 mg/100 ml to about 3.0 mg/100 ml.
Claim 4
Dependent Claim
14 words
The method of claim 2 wherein said concentration does not exceed 2.0 ml/100 ml.
Claim 5
Dependent Claim
13 words
The method of claim 3 wherein said concentration is about 1 mg/100 ml.
Claim 6
Dependent Claim
18 words
The method of claim 1 wherein said sphering agent is an alkali metal salt of an alkyl sulfate.
Claim 7
Dependent Claim
14 words
The method of claim 5 wherein said alkyl sulfate salt is sodium dodecyl sulfate.
Claim 8
Dependent Claim
21 words
The method of claim 1 wherein the concentration of said fixing agent is from about 0.05 w/w% to about 0.3 w/w%.
Claim 9
Dependent Claim
12 words
The method of claim 7 wherein said concentration is about 0.2 w/w%.
Claim 10
Dependent Claim
11 words
The method of claim 1 wherein said fixing agent is glutaraldehyde.
Claim 11
Dependent Claim
13 words
The method of claim 1 wherein said aqueous reagent includes a phosphate buffer.
Claim 12
Dependent Claim
21 words
The method of claim 10 wherein the phosphate buffer is adjusted to provide a reagent pH of from 6.0 to 7.5.
Claim 13
Dependent Claim
21 words
The method of claim 11 wherein said phosphate buffer concentration is about 0.035M and provides a reagent pH of about 7.3.
Claim 14
Dependent Claim
12 words
The method of claim 1 wherein said aqueous reagent includes sodium chloride.
Claim 15
Dependent Claim
21 words
The method of claim 13 wherein the sodium chloride concentration is adjusted to provide a reagent osmolarity to about 290 Os/kg.
Claim 16
Dependent Claim
16 words
The method of claim 1 wherein said temperature is maintained at about 25° C. (room temperature).
Claim 17
Dependent Claim
13 words
The method of claim 1 wherein said reaction period is about 60 seconds.
Claim 18
Dependent Claim
16 words
The method of claim 1 wherein said whole blood dilution factor is about 1:600 to 1:700.
Claim 19
Dependent Claim
54 words
The method of claim 1 wherein said aqueous reagent comprises sodium dodecyl sulfate at a concentration of about 1 mg %, glutaraldehyde at a concentration of about 0.2%, about pH 7.3 phosphate buffer at a concentration of about 0.035M and sodium chloride in an amount to provide a reagent osmolarity to about 290 mOs/kg.
Claim 20
Dependent Claim
31 words
The method of claim 1 wherein the resulting, diluted sample is subjected to light scattering measurement for determination of at least one of red blood cell volumes and cell hemoglobin content.
Claim 21
Dependent Claim
16 words
The method of claim 1 wherein the resulting, diluted sample is measured with a flow cytometer.
References Cited (Backward Citations)
US4389490A
Cited
US4412004A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
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| 3.5 Years (Year 4) | 1989-03-10 | 1989-09-09 | 1990-03-11 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1993-03-10 | 1993-09-09 | 1994-03-11 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1997-03-10 | 1997-09-09 | 1998-03-11 | $7700.00 | Window Closed / Lapsed |