Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
1996-06-21
2001-03-06
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S040500, C435S002000
Reexamination Certificate
active
06197540
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to the preparation and stabilisation of cells, and more particularly to a novel method for preparing and stabilising cells and cell suspensions, especially whole blood and constituents thereof and to the use of novel stabilised cell preparations in the quality control of analytical techniques such as UV microscopy and flow cytometric leucocyte immunophenotyping techniques, immobilised antigen/antibody detection systems and haematology analysers, and blood monitoring techniques such as zinc protoporphyrin (ZPP), red cell folate and blood glucose measurements.
UV microscopy and flow cytometry are techniques used in the diagnosis of haematological malignancies. They are also used to monitor the progress of patients infected with the Human Immunodeficiency Virus (HIV), whether asymptomatic or suffering from ARC or full-blown AIDS. Quality control (QC) of these two techniques is extremely important to arrive at the correct diagnosis and to monitor effective therapeutic regimes. The current QC methods use freshly drawn blood or microspheres coated with a fluorochrome.
The use of fresh blood on a daily basis fails to provide the information on day-to-day variation of the technique or equipment. Furthermore, fluorochrome coated microspheres, though providing a day-to-day monitor of the flow cytometer's performance, cannot be used for UV microscopy work. In addition, they cannot be used to provide quality control for the labelling techniques of leucocytes.
Fixation of normal leucocytes utilising compounds such as aldehydes, although giving stability for 5-7 days, increases cellular autofluorescence. This makes the preparation unsuitable for use as a long-term quality control material. Furthermore, the lysis of red cells by the whole blood lysing technique requires a preparation that will quality control this procedure. The current methods of fixing the leucocytes inhibit this lysing procedure, resulting in a significant increase in debris that interferes with the tests.
In International Application No. PCT/US91/03236, the entire disclosure of which is incorporated herein by reference, there is described a blood diluent and lysing agent for differential determination of white blood cells (leucocytes) in which the stabilising agent is diazolidinyl urea. Such leucocyte preparations have not been suggested as quality control preparations, possibly because they have insufficient stability and lack certain specific antigenic activity for those routine quality control procedures which need to assess results from a large number of laboratories.
International Application No. PCT/US92/03758, the entire disclosure of which is incorporated herein by reference, discloses the use of diazolidinyl urea, imidazolidinyl urea, dimethylol-5, 5-dimethylhydantion, dimethylol urea, 2-bromo-2-nitropropane-1, 3-diol and quaternary adamantane as tissue fixatives which are free of aldehydes. The formulations may inter alia mordants such as zinc, strontium, calcium, barium and chromium salts. It is not suggested, however, that any of these salts have stabilising properties.
Other QC equipment requiring the use of whole blood samples or blood products for calibration include haematology analysers, where currently fixed bovine blood or blood from donkeys and turkeys is used because a suitable source of stabilised human blood is not available. Zinc protoporphyrin (ZPP) and red cell folate monitoring techniques also require a fresh suspension of red blood cells for calibration, again because a suitable stabilised source is not available. Finally the lack of a stabilised source of whole human blood for calibration purposes limits the possibility for diabetics to carry out blood sugar monitoring at home.
It will be appreciated from the above that there is a need for an improved method for stabilising cells, particularly of whole blood and blood products, for a variety of quality control, monitoring and calibration applications.
OBJECTS OF THE INVENTION
It is an object of the invention to provide an improved stabilised cell preparation and an improved method for stabilising cells.
It is also an object of the invention to provide a stabilised whole blood preparation and a method of manufacturing such a preparation.
It is a further object of the invention to provide an improved lysing procedure for the separation of leucocytes from red blood cells in whole blood.
It is a still further object of the invention to provide stable quality control materials which can be used in a wide spectrum of quality control, analysis and monitoring techniques.
SUMMARY OF THE INVENTION
It has now been discovered that a wide range of cells can be stabilised by the addition of a compound comprising a heavy metal to the cells in an effective amount, and that such stabilised cells will remain active for much longer periods than those known hitherto.
Thus in one aspect the invention provides a stabilised cell preparation in which the stabilising agent comprises an effective amount of a heavy metal compound.
In another aspect the invention provides a method of stabilising cells by adding thereto an effective amount of a stabilising agent comprising a heavy metal compound.
The invention is particularly applicable to the stabilisation of whole blood and of blood products and will be henceforth more particularly described with reference thereto. It is to be understood, however, that the invention is not limited to the stabilisation of such materials and is broadly applicable to a wide range of cellular materials and particularly cell suspensions.
In a further aspect, therefore, the invention provides a stabilised whole blood preparation in which the stabilising agent comprises an effective amount of a heavy metal compound and a method of stabilising a whole blood preparation by adding an effective amount of the compound thereto.
The invention also provides a method of stabilising the cellular constituents of whole blood, in particular leucocyte preparations formed for example from lysed whole blood, by the addition thereto of an effective amount of a heavy metal compound.
Thus, in a still further aspect, the invention provides a stabilised leucocyte preparation in which the stabilising agent comprises an effective amount of a heavy metal compound, and a method of stabilising a leucocyte preparation by the addition thereto of an effective amount of a stabilising agent comprising a heavy metal compound.
The stabilised leucocyte preparation of this aspect of the invention can be added to leucocyte depleted whole blood (red blood cells) to form a stabilised whole blood preparation, and the invention accordingly includes in a yet further aspect a stabilised whole blood preparation comprising leucocytes stabilised by the addition thereto of an effective amount of a stabilising agent, and a method of manufacturing a stabilised whole blood preparation which comprises adding a stabilised leucocyte preparation to leucocyte depleted whole blood.
In another aspect the invention provides a stabilised whole blood preparation which comprises stabilised leucocytes and untreated red blood cells.
In still another aspect the invention provides a novel stabilising agent for cellular materials, especially blood and blood products, which comprises an aqueous solution of a heavy metal compound and a formaldehyde.
In yet another aspect of the invention there is provided a lysing agent for the differentiation of leucocytes which comprises a solution of an ammonium or quaternary ammonium salt and a urea, the concentration of urea and the pH of the solution being sufficient to inhibit the monocytes from down-regulating the CD14 antigen sufficiently to allow their detection by immunological means.
REFERENCES:
patent: 4045488 (1977-08-01), Sarges
patent: 4123384 (1978-10-01), Hundt et al.
patent: 4152208 (1979-05-01), Guirgis
patent: 4302355 (1981-11-01), Turner, Jr. et al.
patent: 4324687 (1982-04-01), Louderback et al.
patent: 4704352 (1987-11-01), Miripol et al.
patent: 4806343 (1989-02-01), Carpenter et al.
patent:
Barnett David
Fay Shawn P.
Granger Vivian
Mayr Petra S. M.
Reilly John T.
Northern General Hospital N.H.S. Trust
Saucier Sandra E.
Sheldon & Mak
LandOfFree
Preparation and stabilization of cells using aged transition... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Preparation and stabilization of cells using aged transition..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Preparation and stabilization of cells using aged transition... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2533990