AU629174B2

AU629174B2 - A device and a method for removing blood cells from body fluids which contain erythrocytes, and the use thereof

Info

Publication number: AU629174B2
Application number: AU21654/88A
Authority: AU
Inventors: Heinz-Jurgen Friesen; Henning Hachmann
Original assignee: Behringwerke AG
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1987-08-31
Filing date: 1988-08-30
Publication date: 1992-10-01
Anticipated expiration: 2008-08-30
Also published as: EP0305803A2; AU2165488A; EP0305803A3; JPS6472065A; DE3729001A1

Description

6291 7or4 COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class rnt. Class Application Number: Lodged: 0 tomplete Specification Lodged: Accepted: '7'Virity: 0 Published: FRelated Art: 0 0 Name of Applicant: 00 ee Address of Applicant: n000 Actual inventor: BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany HEINZ-JUGEN FRIESEN and HENNING HACHMANN EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Address for Service: Complete Specification for the invention entitled: A DEVICE AND A METHOD FOR REMOVING BLOOD CELLS FROM BODY FLUIDS WHICH CONTAIN ERYTHROCYTES, AND THE USE THEREOF The following statement is a full description of this invention, including the best method of performing it known to US 1.

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-la- BEHRINGWERKE AKTIENGESELLSCHAFT 87/B 032 Ma 610 Dr. Ha/Bn A device and a method for removing blood cells from body fluids which contain erythrocytes, and the use thereof The invention relates to filters and the use thereof, and to a method for removing blood cells from body fluids which contain erythrocytes.

The most important sample material in investigations in clinical chemistry is plasma or serum obtained from whole blood. It is often impossible to use whole blood directly because, in particular, the erythrocytes interfere with the tests owing to their intrinsic color or as particles. This 10 is why blood cells are removed by centrifugation. This is the method of choice when carrying out many different analyses on a blood sample. There is a demand, in emergency diagnosis or in individual tests, for more rapid methods for

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obtaining serum or plasma as well as for test methods in 15 which smaller blood volumes can be used.

EP-A 0,143,574 discloses the removal of erythrocytes from whole blood by adding erythrocyte-agglutinating substances, such as antibodies against erythrocytes or lectins, in S20 solution, and removing the agglutinate.

EP-A 0,045,476 describes the use of fiberglass filters for obtaining plasma or serum from whole blood.

German Patent 3,508,427 describes the obtaining of plasma or serum from whole blood by use of an absorbent matrix impregnated with a lectin.

EP-A 0,223,978 describes devices and methods for determining blood groups by use of porous materials to which essentially pure antibodies directed against antigenic determinants of the blood groups of erythrocytes are bound.

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L 1: It has been found that filter elements which have been impregnated with a solution of antibodies and, where appropriate, lectins which are able to agglutinate erythtocytes, which elements have been dried, retain all blood tissue when whole blood or a body fluid containing 3rythrocytes is applied. The filtrate obtained from this is cell-free plasma, serum or CSF. Fiberglass-containing filters which have been impregnated with a solution of erythrocyte agglutinating lectin and then dried likewise showed good separation of the cell-free body fluid from the blood tissue.

Hence the invention relates to a device for removing blood cells from a body fluid by use of a fiber-containing filter layer, which comprises the filter layer containing antibodies which agglutinate all erythrocytes and, where appropriate, containing a lectin. The fiber may be synthetic polymer fibers for example cellulox or fiberglass or a mixture of different fibers.

15 The invention also relates to a device for removing blood cells from a body fluid by use of i~i a fiber-containing filter layer, which comprises the filter layer containing fiberglass and a lectin.

The invention additionally relates to a method for producing a device composed, at the ieast, of a fiber-containing filter layer which contains erythrocyte-agglutinating *antibodies and, where appropriate, a lectin, which comprises the filter layer being i impregnated with an aqueous solution of the antibody and, where appropriate, of the lectin, and the impregnated filter layer being dried.

i 25 The invention furthermore relates to a method for producing a device containing a fiberglass-containing filter layer and a lectin, which comprises the filter layer being impregnated with an aqueous solution of a lectin, and the impregnated filter layer being S"dried.

The invention furthermore relates to a method for obtaining tissue-free body fluid by applying an erythrocyte-containing body fluid to a device of this type, and Al ~AJT 1

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3 allowing the tissue-free body fluid to run out as filtrate, or to be absorbed by an absorbent material which is connected to the device.

The invention additionally relates to the use of a device of this type in a diagnostic element for the detection and for the determination of a component contained in the tissuefree body fluid.

Suitable materials for components of an antibody-containing device are cellulose fibers, chemically derivatized cellulose fibers, fiberglass or quartz fibers or other mineral fibers, and natural polymeric fibers whose basic components differ from cellulose. The said fibers can form the fibrous component of the filter layer as individual fibers or as combination, with both a woven and a non-woven struc- .ture being suitable.

.3 ii 91 i *20 0 0 *25 *see of go 6 Suitable antibodies are those which agglutinate erythrocytes. They are preferably obtained by immunization of animals with a mixture of erythrocytes. It is possible to use mono- orolyclonal antibodies.

Lectins which are, where appropriate, added to the impregnation solution are those which likewise agglutinate erythrocytes or those which react with the antibody.

The antibodies and, where appropriate, the lectins are preferably dried directly onto the fiber-containing filter layer from a buffered or unbuffered aqueous solution.

The concentrations of antibodies and, where appropriate, lectins in this solution, which is also called impregnation solution, are suited to the desired erythrocyte-retaining effect of the fiber-containing filter layer. When fiberglass filters are used, a preferred concentration of antibody and, where appropriate, lectin corresponds to at least 5 times the concentration sufficient for complete agglutination.

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4 If the device comprises a filter with fiberglass and lectin but no antibodies as components, it is produced by impregnating a fibergLass-containing filter with a solution of one or more lectins, and then drying it. This entails the use of a solution of a Lectin or of a mixture of lectins which agglutinate all human erythrocytes. The Lectins from Solanum tuberosum (potato) or from Lycopesicon esculentum (tomato) are preferred as the singLe Lectin or in combination with other Lectins. The lectin is present in the impregnation soLution in a preferred concentration of 1-20 mg/L. 5 mg/L are particuLarLy preferred.

°,p It is also possible to add to the impregnation soLutions proteins and detergents which Limit the binding of analyte '15 or antibodies to the surface. The concentrations of these additions are chosen for this such that inhibition of hem- S aggLutination is avoided. Furthermore, the concentration of the detergents is Limited in such a way that they do not S bring about any hemolysis. The minimaL detergent concentra- 20 tion is adjusted to ensure optimal wettabiLity of the filter for the test.

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SuitabLe proteins are Polygeline, serum albumin, geLatin, ovaLbumin or casein. The preferred concentration range for .25 this is 0.5 10 g/l, particuLarly preferabLy 0.8 1.2 g/l.

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Suitable detergents are Zwittergent 3-10 (Calbiochem), RDapraL 205 (AKZO) or Rpluronic F-68 (Atlas Chemie) in a concentration of 0.1 5 g/L and, especiaLLy, of 0.1 0.2 g/L. Particularly suitable are Rzwittergent 3-08 and RDapraL 205 in a concentration of 0.1 5 g/L, with 0.1 0.2 g/L being preferred.

The pH of the impregnation solution is preferably in the range pH 6 8 particularly preferably at pH 7.0 If the device is contained in a diagnostic element arranged in the form of a layer or in the form of a sheet and is connected by one surface to an absorbent layer, once the ^IgS

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5 erythrocyte-containing body fluid has been applied to Ianother surface of the device, which is not connected to j an absorbent layer, the tissue-free fluid is absorbed by the absorbent Layer by capiLLary forces and is thus availabLe for the detection and for the determination of a component contained therein.

Ii Examples K 10 Preparation of erythrocyte-agglutinating antibodies S i Rabbits were immunized with a pool of washed erythrocytes from 30 donors comprising equal proportions of the four blood groups 0, A, B and AB. Agglutinating antibodies were measured in serial dilutions of the serum in phosphate- Sbuffered saline (PBS) by incubating 50 pl of diluted serum, 50 4l of PBS and 25 pl of 20 ml/l erythrocyte suspension in PBS in the wells of microassay plates at 370C for 60 min, reading after a few hours for carpet and button formation, i 20 and recording as titer the dilution at which the transition from carpet to button formation was seen. Titers .i *between 640 and 1280 were obtained with erythrocytes of blood groups 0, A, B and AB.

25 The immunoglobulin G (IgG) fraction was obtained from the serum by chromatography on DEAE-cellulose and, after adjustment to 40 mg/I protein, had an agglutinating antibody titer of 2560 with erythrocytes of each of the blood groups mentioned. This preparation is called Ery Ab hereinafter.

Solutions of Ery Ab Aqueous solutions of 5 pg to 22 g/l Ery Ab, to which 1 g/L RPolygeline and 0.1 g/ RZwittergent had also been added, were prepared and adjusted to pH 7.2.

Solutions of Lectins Lectin from Solanum tuberosum or from Lycopersicon

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escuLentum was dissolved in S R PoLygeline and 0.1 g/L Zwi jg/L, and the solution obt to pH 7.2.

6 an aqueous solution of 1 g/L ttergent in concentrations of ained in this way was adjusted Fiber-containing filters 060

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0 @5 Pieces 6 x 6 mm or 7 x 7 mm in size were cut out of a) No. 2668/8 paper from Schleicher SchuLL, Dassel, or b) No. 030-PG fiberglass filters from Pall, Dreieich (FRG), from which the backing fabric had been removed, or c) Type A fiberglass filter from Whatmann, or d) Extra Thick fiberglass filter from Gelman, or e) QMA quartz fiber filter from Gelman.

Production of a device according to the invention a) Devices for methods with paper as absorbent substrate 20 The fiber-containing filters were impregnated with 8 or 38 or 40 vl/0.

3 6 cm 2 (appropriate for the volume absorbed by the particular filter) solution of serum albumin or Ery Ab 4, 6, 8 and 22 mg/ml), and the filters were then dried at 50 0 C in a circulating-air drying oven for 20 min. The type A fiberglass filter was impregnated with the volume absorbed by the filter using serial 10-fold dilutions of the antibodies from 0.5 g/l to 50 jg/L.

b) Devices for methods with agarose-containing film as absorbent layer Relatively large pieces of filter were impregnated with the impregnation solutions indicated above. Excess impregnation solution was removed by blotting with filter paper.

The amount of fluid remaining in the filter corresponded to 80 90 of the volume absorbed. The filters were then dried at 30 0 C and at 30 torr in a vacuum drying oven for 6 hours.

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7 Use of a device according to the invention Design of experiment: a) Experiments with paper as plasma-absorbing substrate: Devices 6 x 6 mm in size, produced by impregnation of the described fiber-containing filters with Ery Ab, had one end placed on No. 2040a filter paper from Schleicher Schull, Dassel. The 2040a paper was fixed on a substrate using double-sidedRTesa adhesive tape from Beiersdorf. For the purpose of comparison, non-impregnated fiber-containing filters k*ere also placed on the No. 2040a paper.

b) Examples with agarose-containing film Layer Devices 7 x 7 mm in size, produced by impregnation of the described fiber-containing filters with Ery Ab, and a piece of polystyrene film 7 x 11 mm in size, which had been coated with 18 g of agarose and 6 g of titanium dioxide/m (volume absorbed: 7 pl), were placed side by side on a cellulose acetate propionate substrate which was 7 x 20 mm in size and had on its surface 20 longitudinal grooves 0.125 mm deep.

Moreover, the agarose-coated side of the film was in con- 25 tact with the side of the substrate with the grooves.

S* Experimental procedure: a) Absorption of plasma by paper S il drops of heparinized blood were placed on the device with No. 2668/8 fiber-containing filter, and 15 il drops of heparinized blood were placed on the device with No. 030-PG fiberglass filter. The diffusion of the fluid through the devices into the underlying No. 2040a paper was observed.

b) Absorption of plasma by a film layer In this case, 65 il drops of heparinized blood were placed Io 8 8 i on the devices. The times for the qrooves covered by the film to be filled, and the contamination of the plasma with erythrocytes, were recorded.

Results: i a) Experiments with a device containing filter paper, paper as plasma-absorbing agent, and blood of blood group B: Where the filter paper was non-impregnated or impregnated with serum albumin, the filtrate which migrated into the underlying agent was deep red in color and contained erythrocytes. The red coloration migrated with the fluid front. With the devices according to the invention.. the red coloration migrated no more than half as far as the i fluid front. With the devices which had been impregnated with solutions of 8 and 22 g/L Ery Ab, no red coloration emerged from the device.

O o S b) Experiments with devices containing 030-PG fiberglass filters, paper as plasma-absorbing agent, and blood of blood group B: Where the fiberglass filter had not been impregnated beforehand, a deep red coloration migrated out of the filter with the fluid front. Where the filter was impregnated with serum albumin, the red coloration migrated out of the filter about 2/3 as far as the front. With the filters impregnated with Ery Ab, that is to say the devices according to the invention, no red coloration now emerged from |the filter in any case. The fluid absorbed by the underlying filter had the color of a non-hemolytic serum or plasma.

c) Experiments with a device containing GF/B fiberglass filter, paper as plasma-absorbing agent, and blood of unknown blood group: Where the filter had not been impregnated beforehand, a 9 red cloud migrated out of the filter when 70 IL of whole blood was applied thereto.

This cloud was not observed when antibody concentrations down to 5 mg/l were impregnated beforehand.

This cloud was likewise not observed when Lectin from Solanum tuberosum or from Lycopersicon esculentum was impregnated beforehand in concentrations down to 5 mg/l.

d) Experiments with devices containing Extra Thick fiberglass filters or QMA quartz fiber filters, agarose film as plasma-absorbing agent, and blood of various blood groups: 15 1 When the erythrocytes were completely removed in the device, O* no erythrocytes were detectable in the grooves in front of the white background of the film (test result: to Where the test results were poor at Least some grooves 20 which contained plasma contaminated with erythrocytes were 5e detectable as red stripes. The test result was good with devices which contained quartz fiber or fiberglass and had S" been impregnated with solutions of Ery Ab in a concentration greater than 0.1 g/l or with Lectin in a concentration 25 greater than 5 mg/L.

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The test result is summarized below.

Filter Time until the Test result material test element is Without With With filled with plasma impregnation Ery Ab lectin Quartz fiber 96 102 sec (n 4) Fiberglass 15 41 sec (n 4) 10 e) Use of the device in a diagnostic element for the determination of glucose in whole blood: To produce a means for the determination of glucose in whole blood, the Rapignost R glucose test section of the urinary glucose test strip from Behringwerke, with dimensions of 6 x 15 mm, was fixed to a substrate with doublesided Tesa adhesive tape from Beiersdorf. A Pall 030-PG fiberglass filter (dimensions: 6 x 6 mm) from which the backing fabric had been removed as previously and which had been impregnated with anti-Ery in a concentration of 2 mg/ ml had one end placed on the RAPIGNOST test paper. 15 il drops of whole blood with different glucose contents were placed on the fiberglass filter. The coloration forming .15 in the test paper was observed. A blue-green coloration formed, within 1 2 min, in the test paper wetted with the plasma emerging from the filter. The intensity of this corresponded to that obtained when, in place of whole blood, centrifuged plasma or serum obtained from the same blood S 20 samples was used. The same results were obtained when a correspondingly impregnated Gelman type A fiberglass filter was used. With 2668/8 paper, reading was possible in the region of the test paper in which the plasma was separated from the erythrocytes.

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Claims

1. A device for removing erythrocytes from a body fluid, when said body fluid is applied to said device comprising a fiber-containing filter layer said filter layer containing antibodies against erythrc .ytes contained in said body fluid, the volume of said filter layer being smaller than the volume of a sample applied; and (ii) an absorbent layer in contact with said filter layer being capable of absorbing the entire liquid volume of the erythrocyte-free body fluid and being capable of use as a diagnostic medium.

2. A device according to claim 1 wherein said antibodies are monoclonal antibodies.

3. A device according to claim 1 or 2 wherein said filter layer additionally contains a lectin or a mixture of lectins.

4. A device as claimed in claim 1, wherein the filter layer contains natural polymeric fibers, preferably cellulose fibers, chemically derivatized cellulose fibers, fiberglass or other mineral fibers, synthetic polymer fibers as single fiber component, or a mixture of several fiber components. A method for producing a device composed of a fiber-containing filter layer which contains erythrocyte-agglutinating antibodies as claimed in claim 1 which comprises the filter layer being impregnated with an aqueous solution of the antibody, said antibody being applied in a volume greater than the volume of the filter layer, and the impregnated filter layer being dried. G. A method for producing a device composed of a fiber-containing filter layer which contains erythrocyte-agglutinating antibodies and lectins as claimed in claim 3, which comprises the filter layer being impregnated with an aqueous solution of the antibody and the lectin, and the impregnated filter layer being dried. 1-^s

7. A method for obtaining erythrocyte-free body fluid, in which erythrocyte- containing body fluid is applied to a device as claimed in any one of claims 1 to 3, and the erythrocyte-free body fluid is obtained as filtrate from said fiber-containing filter layer.

8. A method for obtaining erythrocyte-free body fluid, in which erythrocyte- containing body fluid is brought into contact with a device as claimed in any one of claims 1 to 3, and then erythrocyte-free body fluid is extracted from said absorbent layer.

9. The use of a device as claimed in any one of claims 1 to 2 in diagnosis for the presence of a component contained in the erythrocyte-free body fluid. DATED this 20th day of July, 1992. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRAUA DBM:KJS:JZ (Doc. 14) AU2165488.WPC et o* o•f~f' o