WO1992019966A1 - Temoin d'hematologie pour globules blancs - Google Patents
Temoin d'hematologie pour globules blancs Download PDFInfo
- Publication number
- WO1992019966A1 WO1992019966A1 PCT/US1992/003772 US9203772W WO9219966A1 WO 1992019966 A1 WO1992019966 A1 WO 1992019966A1 US 9203772 W US9203772 W US 9203772W WO 9219966 A1 WO9219966 A1 WO 9219966A1
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- WIPO (PCT)
- Prior art keywords
- lipoprotein
- reference control
- white blood
- blood cells
- mixture
- Prior art date
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2496/00—Reference solutions for assays of biological material
- G01N2496/05—Reference solutions for assays of biological material containing blood cells or plasma
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
Definitions
- the present invention relates to a method for preparing a hematology reference control and calibrator for hematology instruments used to measure granulocytes, lymphocytes, monocytes, eosinophiles and basophiles.
- hematology instruments which can analyze blood to determine the number of granulocytes, monocytes, lymphocytes, eosinophiles and basophiles. These instruments include the Coulter STKS, Unipath 3000, TOA NE 8000, Technicon H-l and Technicon H-6000. Because these instruments employ different methods to measure the five populations of white cells, it has been necessary to employ a different type of reference control with each instrument. This fact has necessitated that the user stock separate controls for each of the types of instruments. From the user's standpoint, this situation is not only inconvenient, but can be expensive as well.
- the present invention provides a reference control for hematology instruments comprising: i) white blood cells fixed with an aldehyde fixing agent suspended in an isotonic aqueous suspension medium; and ii) a composition comprising at least one lipoprotein in an amount sufficient to provide a mixture that gives a white blood cell histogram profile that is substantially the same as that of whole blood.
- the aldehyde fixative comprises a mixture of formaldehyde and glutaraldehyde in a ratio of 3 to 20 ml formaldehyde: 0.01 to 1 ml of glutaraldehyde per 100 ml of fixing solution.
- the use of a mixture of formaldehyde and a small amount of glutaraldehyde reduces the amount of lipoprotein necessary to provide a histogram profile for fixed or stabilized white blood cell controls that is substantially the same as that of whole blood.
- Particularly preferred reference controls of the invention further include predetermined amounts of both stabilized red blood cells and simulated blood platelets.
- the reference control includes stabilized red blood cells
- antioxidants are ascorbic acid, butylated hydroxytoluene, Probucol (acetone bis (3, 5-di-test-butyl-4-hydroxyphenyl) mercaptole) bisphenabid, Rutin (Rutoside, quercetin-3-rutinoside) , alpha-tocopherol, Deferoxamine Mesylate and Sulfasalazine.
- antioxidants employed will vary depending principally upon the particular antioxidant selected and the amount of lipoprotein employed. In general, however, the amount ranges from 0.1 to 100 mg/d£. More specifically, the following table sets forth amounts of the recited antioxidants that are normally employed: ANTIOXIDANTS USED WITH LIPOPROTEINS
- Probucol acetone bis (3,5- di-tert-butyl-4-hydroxyphenyl) mercaptole) bisphenabid 0.1- 10 mg/d£ 3 mg/d£
- Rutin (Rutoside, quercetin-3- rutinoside) 0.001-0.01 0.01 mg/d£ mg/d£
- the invention also provides a method of preparing a stabilized reference control for hematology instruments comprising mixing white blood cells fixed with an aldehyde fixing agent, an isotonic aqueous suspension medium and at least one lipoprotein in an amount sufficient to provide a mixture that gives a white blood cell histogram profile that is substantially the same as that of blood.
- stabilized red blood cells and simulated platelets are mixed with the isotonic aqueous suspension, lipoprotein and an antioxidant to inhibit lysis of the stabilized red blood by the lipoprotein.
- Figure 1 illustrates a white blood cell histogram for whole blood. It is a goal of the invention to make a reference control which produces a histogram like that of Figure 1.
- Figure 2 is a white cell histogram made after washing the white cell component of Figure 1 in an isotonic saline solution. This demonstrates the changes to the histogram of Figure 1 caused by washing.
- FIG 3 shows that the histogram for washed white cells (as in Figure 2) will look more like Figure 1 after treatment with concentrated serum.
- Figure 4 shows that the histogram for washed white cells (as in Figure 2) will look more like Figure 1 after treatment with concentrated plasma solution.
- Figure 5 illustrates the effect on the histogram for washed and fixed white cells showing the variation from Figure 1.
- Figure 6 shows the histogram for the reference control of the invention. It illustrates the effectiveness of the invention in producing a histogram similar to Figure 1.
- the reference control for the invention provides a 5-part differential on the TOA NE-8000, Technicon H-l, Technicon H-6000, Cel-Dyn 3000 as well as the Coulter STKS. DETAILED DESCRIPTION OF THE INVENTION When fresh whole blood samples are collected, they contain five different white cell subpopulations, four of which can be seen in the histograms in Figures 1 and 2 obtained using a Coulter STKS. The four visible populations are monocytes 10, neutrophiles 12, eosinophiles 14 and lymphocytes 16.
- the lipoproteins suitable for use in the present invention can be either high or low density lipoproteins, both of which are available from a number of commercial sources. Lipoproteins from the serum of various animals, (e.g. horse and bovine) and egg yolk lipoprotein all have been found to exhibit the desired effect. (See Fig. 6) It is also possible to use compositions containing lipoproteins as the-lipoprotein source. Examples of such lipoprotein sources are animal (include man) blood serums and plasmas. (See Fig. 3 and Fig. 4.) Where such lipoprotein-containing compositions are employed, the amount of composition employed in the reference controls of the invention will depend upon the concentration of the lipoprotein. Preferred blood plasma or serum concentrates are those concentrated two to three fold so as to contain about 14 to 17% by weight of protein.
- the amount of lipoprotein added to the stabilized white blood cells is that which effectively moves the white blood cell subpopulations in the histogram to the correct position, i.e. so that the histogram substantially mimics the white blood cell histogram for whole blood.
- the amount of lipoprotein in the final product will vary from about 0.5 to 8.0% by volume, depending upon the type of lipoprotein used. For example, two to three times more low density protein is required than high density lipoproteins. When egg lipoproteins are used about 30% more is necessary than high density lipoprotein.
- the aqueous suspension medium employed in the reference control of the invention is one that is non-deleterious to the white blood cells and, optionally, red blood cells or platelets to be counted.
- the suspension medium is preferably a physiological salt solution such as an isotonic salt solution, advantageously buffered to a pH neutral to alkaline, preferably a pH of 7 to 9.
- Preferred buffering agents include alkali metal phosphates such as disodium phosphate, monopotassium phosphate, monosodium phosphate, sodium citrate and the like and mixtures thereof.
- the white blood cells of the reference control of the invention are fixed with an aldehyde fixing agent to partially rigidify their cell membranes and render them stable.
- Aldehyde fixing agents for white blood cells are well known and include, for instance, formaldehyde and glutaraldehyde.
- the fixatives are preferably prepared in phosphate buffered saline. The amount of aldehyde employed in the fixatives will vary depending upon the particular aldehyde employed but normally falls in the range of about 1 to 20% by volume of fixing agent.
- the preferred fixative is a mixture of formaldehyde and glutaraldehyde in a ratio of 0.01 to 1.0 ml of glutaraldehyde: 3 to 20 ml formaldehyde per 100 ml of fixative. It has been discovered that reference controls made up of white blood cells fixed with the mixture of formaldehyde and glutaraldehyde require less lipoprotein to properly position the white blood cells. The presence of a small amount of glutaraldehyde decreases the amount of lipoprotein required.
- the reference control of the invention may also include stabilized red blood cells and simulated blood platelets commonly employed in prior art reference controls in a number, size, and distribution equal to that of human whole blood. Suitable simulated blood platelets are described in U.S. Patent Nos. 4,436,821 and 4,160,644 hereby incorporated by reference.
- the red blood cells can be stabilized by treatment with aldehyde prior to suspension in the isotonic suspension medium or alternatively by incorporating stabilizing additives in the isotonic suspension.
- Illustrative of prior art methods for stabilizing or fixing red blood cells include those described in U.S. Patent Nos. 4,358,394; 4,299,726; 3,873,467; 4,390,632; 4,698,726 and 4,777,139, all hereby incorporated by reference.
- EXAMPLE 1 Human white blood cells are obtained as concentrates from commercial blood banks. The white blood cells are washed and freed of red blood cells by placing in tris-ammonium chloride buffer for several minutes. Tris-ammonium chloride buffer is prepared by adding 7.5 g of ammonium chloride and 20 ml Trizma base (tris (hydroxy methyl) amino methane) to one liter of distilled water. The white blood cells are stabilized by adding phosphate buffered saline containing 13 ml of 37% formaldehyde/100 ml of phosphate buffered saline.
- the cells are held in the fixative for 24 hours and then washed with phosphate buffered saline.
- the white blood cells are added to a red cell preservative diluent containing lipoprotein.
- the amount of lipoprotein varies with the purity and type used.
- the commercial preparation used is Miles Supertrate R and it is used at .5 - 5% v/v. This mixture of blood cells and lipoprotein result in a stable control that produces a histogram similar to whole blood on the Coulter STK-S, TOA NE-8000, Technicon H-l, Technicon H- 6000, and the Cell-Dyn 3000.
- EXAMPLE 2 White blood cells are fixed with 37% formaldehyde diluted to 20% v/v in phosphate buffered saline containing 0.33 ml of 25% glutaraldehyde. After fixing for 24 hours the cells are washed with saline and added to red cells in a stabilizing solution containing sodium citrate, magnesium gluconate and antibiotics. After adding the white blood cells, simulated platelets prepared as described in U.S. Patent No. 4,160,644 are added.
- the simulated platelets and the white blood cells are added to the red cells in the stabilizing diluent at normal, high abnormal and low abnormal levels.
- lipoprotein Supertrate Miles - a high density lipoprotein >70%, protein 5.8, 48-49 dynes/cm 2 ) is added at a concentration of 2% v/v (0.5 - 5.0%) .
- Figure 6 shows a histogram on STKS of the completed product. This product also provides a 5- part differential on the TOA NE-8000, Technicon H- 1, Technicon H-6000, " ' Cell-Dyne 3000 as well as the STKS.
- a mixture of formaldehyde and glutaraldehyde used a fixative is preferred because it decreases the amount of lipoprotein required to produce a white cell histogram which substantially mimics the white cell histogram for whole blood.
- Example 2 is repeated adding the following antioxidants to the stabilizing solution in the designated amounts:
- Rutin (Rutoside, quercetin-3-rutinoside)
- the antioxidant is effective in inhibiting lysis of the red blood cells.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
Témoins de référence composés de globules blancs fixés par aldéhyde présentant un profil d'histogramme de globules blancs correspondant sensiblement à celui du sang total, ces témoins obtenus par les additions d'une lipoprotéine au témoin. Un antioxydant est de préférence inclus afin d'inhiber la lyse par ladite lipoprotéine de globules rouges stabilisés,
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US69733191A | 1991-05-09 | 1991-05-09 | |
US697,331 | 1991-05-09 |
Publications (1)
Publication Number | Publication Date |
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WO1992019966A1 true WO1992019966A1 (fr) | 1992-11-12 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/US1992/003772 WO1992019966A1 (fr) | 1991-05-09 | 1992-05-11 | Temoin d'hematologie pour globules blancs |
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WO (1) | WO1992019966A1 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0685994A1 (fr) * | 1993-02-25 | 1995-12-13 | Abbott Laboratories | Systeme de reactifs polyvalent de lyse rapide d'echantillon de sang total |
CN109932514A (zh) * | 2019-03-26 | 2019-06-25 | 江苏力博医药生物技术股份有限公司 | 血小板醛化试剂的制备及应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4099917A (en) * | 1977-07-21 | 1978-07-11 | Technicon Instruments Corporation | Process for preparing a cell suspension from blood for discrimination of white blood cells and platelets from other blood particles |
US4324687A (en) * | 1979-02-15 | 1982-04-13 | Louderback Allan Lee | Blood biochemistry control standard |
US4425334A (en) * | 1982-04-02 | 1984-01-10 | The Regents Of The University Of California | Functional oxygen transport system |
US4436821A (en) * | 1982-04-12 | 1984-03-13 | Streck Laboratories, Inc. | Simulated human platelets from red blood cells |
-
1992
- 1992-05-11 WO PCT/US1992/003772 patent/WO1992019966A1/fr active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4099917A (en) * | 1977-07-21 | 1978-07-11 | Technicon Instruments Corporation | Process for preparing a cell suspension from blood for discrimination of white blood cells and platelets from other blood particles |
US4324687A (en) * | 1979-02-15 | 1982-04-13 | Louderback Allan Lee | Blood biochemistry control standard |
US4425334A (en) * | 1982-04-02 | 1984-01-10 | The Regents Of The University Of California | Functional oxygen transport system |
US4436821A (en) * | 1982-04-12 | 1984-03-13 | Streck Laboratories, Inc. | Simulated human platelets from red blood cells |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0685994A1 (fr) * | 1993-02-25 | 1995-12-13 | Abbott Laboratories | Systeme de reactifs polyvalent de lyse rapide d'echantillon de sang total |
EP0685994A4 (fr) * | 1993-02-25 | 2001-10-31 | Abbott Lab | Systeme de reactifs polyvalent de lyse rapide d'echantillon de sang total |
CN109932514A (zh) * | 2019-03-26 | 2019-06-25 | 江苏力博医药生物技术股份有限公司 | 血小板醛化试剂的制备及应用 |
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