Classifications

• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54386—Analytical elements
  • G01N33/54387—Immunochromatographic test strips
  • G01N33/54388—Immunochromatographic test strips based on lateral flow
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

Definitions

• the present invention relates to methods for the determination of minor cell populations in heterogeneous cell populations.
• the methods are for enrichment and quantification of the minor cell population in heterogeneous cell populations such as fetomaternal hemorrhage; for the parallel determination of cell-bound analytes and thus for the determination of both cell populations in mixed field reactions after transfusion, in fetomaternal hemorrhage or in so-called chimeras; to detect an analyte present in low concentration on cells; and / or for determining the hematocrit value.
• a device suitable for the method is provided.
• FMH fetal hemorrhage
• the standard dose of anti-D therapy is no longer sufficient protection.
• standard immunization in the USA is 250 to 300 ⁇ g of anti-D (IgG), which results in sufficient prevention in a pregnant woman whose circulation has been transferred to 15 ml of fetal erythrocytes, ie 25 to 30 ml of fetal blood.
• the standard dose administered is often lower, namely 100 to 150 ⁇ g anti-D (IgG), which protects at an FMH of 8-10 mL.
• Any device that provides anti-D prophylaxis must use a method that detects a larger FMH than usual (Issitt PD and Anstee DJ., Applied Blood Group Serology [4 th edition], Montgomery Scientific Publications.) Chapter 41: Hemolytic disease of the newborn, pp. 1045-1050.
• This test is based on the detection of erythrocyte aggregates and their microscopic evaluation. Detection of the fetal D antigen.
• This test is based on the higher resistance of fetal erythrocytes to acid elution and its microscopic evaluation. Detection of fetal cells.
• D antigen or hemoglobin F is labeled with appropriate antibodies and detected via fluorescent second antibody. Detection of fetal D antigen / hemoglobin. Garratty G, Arndt P. Transfusion 1995; 35: 157. Davis bra. Clin Lab Med 2001; 21: 829.
• the test is the first test to determine an FMH based on a routine blood typing method (gel technique). It is based on the consumption of anti-D reagent by fetal erythrocytes. The reaction supernatant is incubated with D-positive test cells and centrifuged in the gel test. Detection of Fetal D Antigen Lapierre Y, Rigal D, Adam J, Josef D, Meyer F, Greber S, Drot C. Transfusion 1990; 30: 109. David M, Stelzer A, Wittmann G, Dudenhausen JW, Salama A. Z Obstetrics Neonatol 1999; 203: 241st
• D antigen Another challenge for donor and recipient serology are weak or partial forms of the D antigen. Thanks to monoclonal antibodies and confirmation in the Indirect Coombs test, the typing of such weak blood groups appeared to be ensured until the DEL phenotype was described, which represents a particularly weak D expression ("normal” D: 10-30,000 antigens per erythrocyte (RBC ) D weak: 400 to 1000, DEL: ⁇ 30).
• DEL can only be detected indirectly with extremely complex adsorption-elution tests comprising up to 10 washes.
• DEL is of relevance for transfusion medicine, as D recipients of a DEL person can train anti-D antibodies. The problem is so worrying that it is currently being discussed in the scientific community, whether the D status of all (serological) D donors should be verified by molecular methods.
• Transfusions should always be ABO same or compatible and D compatible. In certain cases, z. It is clinically indicated, for example, in pre-transfused patients, to transfuse same blood groups for other antigens. However, it is never possible to transfect blood group-identical in all blood groups (exception: autologous transfusion). As a result, after transfusions, the situation regularly arises that a person is diagnostically positive and negative for certain blood group characteristics. In diagnostics, so-called mixed-field reactions are found, as can be well detected by sensitive methods - especially those that can spatially separate individual erythrocytes and hemagglutinates during detection.
• erythrocytes of our example are centrifuged through such an anti-K column, most of the cells will sediment on the bottom because they are K- and a small part will be held on or in the gel (K +), which corresponds to the detection of the above-described mixing field. If such erythrocytes are applied to the MDmulticard (Medion Diagnostics) as above, then a weak band in anti-K is also detectable. However, in contrast to the gel technique, the method disclosed in WO2005005991 can not simultaneously visualize the negatively reacting cell population. All other methods of the prior art have no properties comparable to the gel systems and the MDmulticard for the detection of mixed-field reactions.
• the hematocrit or the total volume of erythrocytes expresses the volume-related proportion of erythrocytes in comparison to the total volume in whole blood in percent.
• the volume of erythrocyte in the whole blood is influenced by the volume and the number of erythrocytes.
• the hematocrit can typically be determined by centrifugation of blood-filled capillaries (Strumia MM, Samble AB, Hart ED, 1954, Am J Clin Path, 24: 1016), by centrifugal displacement setting the proportion of sedimented cellular components in relation to the total volume.
• the hematocrit can be determined using electrical impedance methods.
• the current flowing in an electrolyte solution between anode and cathode current is influenced by particles of other conductivity, which enter the stream.
• the current changes are registered as pulses. From a certain pulse amplitude can be closed on a certain particle size and in hematology so that on a particular cell type, such as red blood cells. From a summation of pulses per volume measured, the cell count and hematocrit can be deduced (Sysmex KX-21N Operator's Manual, 1999).
• the present invention is therefore based on the object of providing simple, cost-effective, rapid results, automatable methods for determining the minimum cell population in heterogeneous populations, such as fetomaternal hemorrhage or so-called chimeras. Furthermore, simple, rapid and sensitive methods for detecting an analyte present in low concentrations on cells, for determining the hematocrit value and / or for the parallel determination of cell-bound analytes in mixed-field reactions are to be provided. Preferably, the methods should have an increased sensitivity to known methods.
• a porous membrane (2) suitable for penetrating cellular components having at least one indicator zone on the membrane which is capable of interacting with the cell-bound analyte, the indicator zone being at least one Contains binding element against the cellulary-bound analyte and at least one absorption region (3) on the membrane, which receives the liquid after passing through the indicator zones,
• the at least one indicator zone lies between the task zone (5) and the absorption zone (3), and wherein the method for enrichment and quantification of the minor cell population in heterogeneous cell populations such as fetomaternal hemorrhage or so-called chimeras, for detecting a low concentration Cells present analyte, for determining the hematocrit value and / or for the parallel determination of cell-bound analytes in mixed field reactions is performed.
• heterogeneous cell populations such as fetomaternal hemorrhage or so-called chimeras
• the method comprises the steps:
• the method according to this preferred embodiment is preferably carried out for enrichment and quantification of the minuscule cell population in heterogeneous cell populations, such as fetomaternal hemorrhage, or for detection of low-concentration analyte analytes.
• This embodiment will hereinafter also be referred to as "incubation method”.
• a lateral flow device is known. It is used for the simultaneous determination of erythrocyte antigens and serum components such as antibodies.
• the lateral flow device known from this document is suitable for the methods specified according to the invention.
• the disclosures of DE 10330982 A1 and WO2005 / 005986 are hereby incorporated in their entirety.
• the lateral flow device is used as a flow cytometer in order to be able to maximally accumulate a second cell population which is present in a small amount in a heterogeneous cell mixture.
• the enrichment is preferably carried out quantitatively by using a maximum amount of whole blood, and qualitatively by an incubation effect caused by volume increase.
• the reading field in the lateral flow device is analogous to the flow cell in the flow cytometer. The background is kept small by washing out the more concentrated cell type and immobilizing the smaller one in the reading window.
• the present method used to determine FMH is highly sensitive and allows the detection of approximately 0, 1% to 0.2% fetal D + cells in maternal blood).
• the device used in the method comprises at least two indicator zones which are arranged one behind the other in the flow direction, so that the sample liquid flows through more than one indicator zone per flow track, the indicator zones containing binding elements against cell-bound analytes
• Method includes: a) applying a blood sample containing erythrocytes to the application zone; b) applying a diluent to the feed zone; c) performing the assay; d) evaluating the assay by determining whether erythrocytes are bound to the indicator zone (s),
• the method is carried out for the parallel determination of cell-bound analytes in fetomaternal hemorrhage (FMH), for the determination in mixed field reactions, for the detection of a homologous transfusion or for the determination of the hematocrit value.
• FMH fetomaternal hemorrhage
• an apparatus for the direct determination of cell-bound analytes in a fluid sample comprising:
• a task zone (5) for applying the liquid sample a porous membrane capable of penetrating cellular components having at least two indicator zones on the membrane which are capable of interacting with the cell-bound analyte, the indicator zones being cell-bound binding elements
• the lateral flow device provided according to the invention differs from that of WO2005 / 005991 in that, in the preferred embodiment shown here, it has two indicator zones lying one behind the other in order to be able to visualize both cell populations simultaneously in a mixed field.
• the sensitivity for the detection of a mixed field is higher than in the only routine method so far that can be used to detect mixing fields, namely the gel technique (DiaMed).
• the sensitivity of the method according to the invention allows the detection of a minor cell population, if their proportion is about 1 - 2% of the total population.
• the method and the device enable highly sensitive detection of mixed-field agglutinations. This utility is also surprising because the skilled person would have believed that the first indicator zone after binding of the analyte-bound cells would act as a diffusion barrier for binding to the second indicator zone.
• the lateral flow device can be used for hematocrit determination as a flow cytometer, which has at least two indicator positions one behind the other to be able to detect the erythrocyte concentration of a blood sample. The higher the concentration, the more points in a line give a positive signal.
• the process can be extended to a 2D array by multiple traces of tile with z. B. each 5 anti-erythrocyte dots are placed side by side, the individual traces of flow have increasing or decreasing anti-red blood cell concentrations.
• the use of the lateral flow device also allows the parallel performance of blood grouping and hematocrit determination.
• FIG. 1 shows a schematic representation of a device suitable for carrying out the incubation method described above.
• the reference signs have the following meaning: (1) carrier layer; (2) porous membrane; (3) absorption area; (4) sealing element; (5) task zone; and (6) indicator zone area.
• FIG. 2 schematically shows a possible application pattern for the detection of mixed field reactions.
• FIG. 3 shows the possible dimensions of the application pattern described in FIG. 2 on a membrane suitable for the detection of mixed-field reactions.
• FIGS. 4A and 4B illustrate the detection of a mixed-field reaction with the aid of the two-indicator zone method according to the invention.
• FIG. 4A shows the result of the examination of a blood sample with 100% A cc D. ee and 0% B CC D. EE.
• Figure 4B shows the result of examining a blood sample with 98% A cc D. ee and 2% B CC D.EE.
• FIG. 5 shows the determination of the hematocrit value with the aid of the method according to the invention.
• heterogeneous cell populations means that one cell population is present in lesser amount or concentration relative to one or more other cell populations that are also present in the heterogeneous cell populations.
• concentration of the minor cell population relative to the entire heterogeneous cell population is thereby less than 50%, preferably less than 10%, more preferably less than 1%, especially less than 0.1%.
• homologous transfusion means transfusions that are compatible in its essential characteristics, the more characteristics that are checked after such a transfusion, the more likely it is to be able to detect via mixing fields that have been transfused, and this probability can be determined by selecting the one to be determined Characteristics are intended to be used in the event of unauthorized blood doping by homologous transfusion.
• autologous transfusion means own blood donation, by which donors and recipients are identical in all characteristics.
• hematocrit means the proportion of erythrocytes [%] in the total blood volume. The following average values are observed: men: 44-52%, women: 37-47%.
• chimera is used for an organism whose cells represent two or more zygotes.
• the membrane of the device used in the invention is a porous membrane.
• Preferred membrane materials are, for example, nitrocellulose (eg UniSart from Sartorius, HiFlow from Millipore, Whatman, AE99 or FF85 / 100 from Whatman Schleicher & Schuell), polyethylene ⁇ lateral FIo from Porex Corporation) or nylon ⁇ Novylon from CUNO) ,
• the membrane has the largest possible pore size, since a high porosity of the membrane, the penetration of particular cellular components of the sample to be determined, for. B. of erythrocytes, favors in the porous structure.
• the device according to the invention is not limited to these properties.
• Preferred are all membranes with a high capillary flow rate, where the capillary flow rate is the time it takes for a dye solution to travel 40 mm on a given membrane.
• Particularly preferred are membranes whose capillary flow rate is ⁇ 100.
• a sealing element is arranged in the flow direction behind the application zone of the device according to the invention on the porous membrane.
• Two- or three-dimensional sealing elements are used, which are placed on the porous membrane and with which a sample application chamber separated from the remaining surface of the porous membrane is used. zone is created.
• the sealing element according to the invention has primarily the effect of a liquid barrier and allows the directional distribution of sample liquid and test reagents in the porous membrane. Furthermore, according to the invention, the sealing element seals off the sample application zone in order to prevent undesired liquid transfer to the other regions of the lateral flow device.
• Preferred embodiments of the sealing element are the web or Trogtial Funnel-shape.
• the shaping of the sealing element takes place by cutting processes from the material used to produce the sealing element.
• the sealing element receives an inner opening
• the preferred embodiments are round, square or rectangular, in the case of the funnel shape to the bottom (membrane contact side) of the sealing element tapered shapes.
• Preferred materials for the sealing element are materials that are not water absorbent (hydrophobic).
• the materials are coated on one side with an adhesive film, for example a pressure-sensitive or self-adhesive acrylate adhesive.
• the sealing element can be bonded directly to the surface of the porous membrane.
• the sealing element may be connected to the lateral flow housing, for example adhesively bonded, wherein in this embodiment the lateral flow housing presses the sealing element onto the surface of the porous membrane and thus the functions of the sealing element are achieved.
• Preferred materials for the formation of two-dimensional density elements are any form of adhesive tapes or adhesive films (eg Tesa 4124 from Beiersdorf AG, ARcare 7815 from Adhesives Research).
• Preferred materials for the formation of three-dimensional sealing elements are flexible, closed-cell elastomeric materials or flexible silicone materials with different material thicknesses, preferably 3-5 mm (eg. Cellular rubber EPDM 140 from Pitzner, silicone rubber or solid rubber, hardness 40 ° or less, from Castan).
• single-piece multiple sealing elements with, for example, 20 individual cavities (trough shape) are arranged on a membrane.
• the device according to the invention is capable of taking up liquid samples containing cells, such as whole blood, without filtering off the cells in the process.
• the sealing element allows the application of large sample volumes on the porous membrane (task zone), without being flooded.
• the sealing element supports the utilization of the receiving properties of the porous membrane.
• the sealing element guarantees a directed sample flow.
• the device according to the invention can work well with or without a sealing element.
• the absorption region (absorption pad) of the device according to the invention mechanically stable materials are preferred, preferably with water absorption capacities of 20-30 g / 100 cm (eg Wicking Paper, Type 300, Whatman Schleicher and Schuell).
• the contact between the absorption pad and the lateral flow membrane of the device according to the invention is produced by pressure and overlapping with the porous membrane. Accurate positioning of the absorbent pad on the membrane is achieved by adhering the absorbent pad to the backing sheet supporting the lateral flow membrane.
• the components of the device according to the invention for the purpose of mechanical reinforcement are applied to a substrate or carrier layer.
• the device according to the invention can function with or without a carrier layer.
• a pressure-sensitive or self-adhesive acrylate adhesive are coated (eg 0.005 "polyester W / GL-187, G & L.)
• the porous membrane and the absorption pad are fixed
• double-sided adhesive backing layer becomes the adhesive second side for fixing the stack on other surfaces, for example, within the lateral flow housing used.
• the device according to the invention is integrated in a housing, whereby the membrane components are pressed against one another and the housing assists the sealing element function.
• the device according to the invention can work just as well with or without housing.
• the method is carried out for the determination of fetomaternal hemorrhage (FMH), wherein the sample contains erythrocytes, wherein the indicator zone preferably contains an antibody or an antibody mixture.
• FMH fetomaternal hemorrhage
• the cell-containing sample may be any sample.
• the cells are preferably blood-borne cells such as erythrocytes, leukocytes or platelets.
• the cells are erythrocytes.
• the erythrocyte-containing sample may in this case be selected from whole blood or blood cell concentrate.
• the blood cell concentrate may be a resuspended erythrocyte sediment.
• the diluent may in principle be any diluent known in the art.
• the diluent is selected from physiological saline, Diluent 1, Diluent 2 (DiaMed), Diluent F (Medion Diagnostics). It is preferably used in the range of 100 ⁇ l to 200 ⁇ l for dilution of the cells.
• the proportion of diluent in the total suspension is preferably lower than that of the whole blood or erythrocyte sediment used in order to effect a relatively high cell concentration and a slow flow of the erythrocytes.
• Performing the assay involves incubating for a sufficient time so that the applied sample migrates from the application zone through the indicator zone (s) to the absorption region.
• Evaluating the assay can be done with the naked eye or automated.
• the indicator zones of the device according to the invention are located on the membrane and comprise binding elements which trap or bind the analytes to be determined in the sample. In the indicator zones, the binding reactions between the analyte and the binding element are detected. As particularly preferred binding elements, antibodies or antibody fragments or lectins are attached to the porous membrane.
• the indicator zones preferably each comprise a binding element against an analyte to be examined.
• the indicator zones can be point-shaped, line-shaped and / or wedge-shaped. Preferably, the line-shaped formation in the direction of flow.
• the minore cell population is detected by a wedge-shaped formation of the indicator zone, which allows better visibility.
• the method is used to determine an analyte present in low concentration on cells carried out, preferably the blood group DEL, and the indicator zone preferably contains an antibody or an antibody mixture.
• the method of the invention may further comprise prior to step a) preparing an erythrocyte sediment by centrifuging the blood sample; Incubating the sediment with a bromelain, papain or ficin solution and resuspending the enzyme-treated sediment include (as described, for example, in AABB Technical Manual, 14th edition, 2003, 693 et seq.). Incubation takes place over a period of 5 to 60 minutes.
• the enzymes to be used are generally available commercially.
• the advantage of the enzymatic pretreatment is a stronger exposure of the D antigens to the erythrocytes and thus a higher sensitivity of the determination.
• step a preferably approximately 100 ⁇ l to 500 ⁇ l blood sample or resuspended erythrocyte sediment are applied in step a). This significantly exceeds the amount of particles and volumes typically applied to lateral flow devices.
• step b preferably about 100 ⁇ l to 200 ⁇ l of diluent are applied in step b).
• the increased total liquid volume leads to a slowed flow, which leads to a quasi incubation of the analyte with the indicator zone.
• the indicator zone contains an anti-D antibody
• this is preferably selected from RUM-I, LDM-3, ESDIM, TH-28, MS-201, MS-26 and LDM-I, as commercially available from Millipore or Alba Bioscience are available.
• antibody mixtures or affinity-purified polyclonal antisera can also be used.
• the method for determining the hematocrit value is carried out, the indicator zone preferably contains an antibody or an antibody mixture, and in step d) the overall pattern in which the erythrocytes are bound to the indicator zones is determined.
• the device comprises at least two indicator zones which are arranged in a flow track and wherein the indicator zones contain identical binding elements against the same cell-bound analyte.
• the indicator zones may be point-shaped, line-shaped and / or wedge-shaped.
• the minore cell population is detected by a wedge-shaped formation of the indicator zone, which allows better visibility.
• the two-indicator zone method allows the representation of the minor cell population adjacent to the major cell population at the proximal location of the indicator zone in relation to the task zone, which is directed against the minor cell population.
• Particularly preferred is an arrangement in which of the two indicator zones in a flow track, the respective proximal line-shaped in the flow direction and the respective distal point is applied.
• the device has at least two traces of flow with at least two indicator zones and the concentration of the binding member increases or decreases from proximal to distal relative to the task zone. This allows a quantitative assessment of the amount of erythrocytes present in the sample in relation to the total volume and thus an approximate determination of the hematocrit.
• the device preferably has at least two traces of flow with at least two indicator zones and the concentration of the antibody spots in a first flow track differs from the concentration in a second flow track.
• the method according to the invention is carried out for the parallel determination of cell-bound analytes in mixed-field reactions or for the detection of a homologous transfusion
• the device in this case contains at least two indicator zones, which are arranged one behind the other in the flow direction, so that the sample liquid per flow track flows through more than one indicator zone, wherein the indicator zones contain binding elements against different cell-bound analytes.
• the cell-bound analyte is selected from a blood group antigen such as A, B, AB, D, C, c, E, e, Cw, K, k, Jka and / or Jkb.
• a blood group antigen such as A, B, AB, D, C, c, E, e, Cw, K, k, Jka and / or Jkb.
• Particularly preferred are the blood group antigens in the form of the reaction pairs A, B; D +, D-; K, k; C, c and / or E, e determined.
• the binding element directed against the analyte is as defined above for the incubation method, preferably selected from an antibody, antibody fragment, lectin, lectin fragment or mixtures thereof.
• an apparatus for performing the two-indicator zone method described above there is provided an apparatus for performing the two-indicator zone method described above.
• test strips in each case 2 different antibodies are applied in series:
• the test strips consist of a central task zone, two indicator zone areas and two absorption areas.
• Millipore HiFlow Plus 075 membranes are cut into strips of 19 x 75 mm (width / length; y / x) for a 10-pair version and glued to a backing layer (backing sheet eg from G & L) , It is operated with a central task zone (bidirectional flow) and in parallel rows on both sides of the task zone in the indicator zone area 0.3 ul lines or 0.1 .mu.l points of solutions of different blood group-specific monoclonal antibodies using a dispenser, eg. B. AD3200 (Biodot), applied, wherein in each case 2 different antibodies, one in each case as a point and a line in series are applied:
• Anti-A clone Burma-1 (Millipore, TL); Anti-B clone LB-2 Millipore, TN); Anti-D clone LDM3 (Alba Bioscience, Z7180100); Anti-C clone MS-24 (Millipore, FFMU, KG); Anti-c clone MS-33 (Millipore, KN); Anti-E clones MS-80 + MS-258 (Millipore, TA); Clones anti-MS-21 + MS-63 (Millipore, FFMU, KL + KQ); Anti-K clone MS-56, (Millipore, KO); anti-k (Alba Clone, Alba Bioscience).
• Anti-RBC (Rabbit IgG Fraction of anti Human RBC, Rockland, 209-4139)
• the dilutions the antibodies are carried out in 15 mM potassium phosphate buffer pH 7.5, 10% (v / v) methanol) as follows: anti-A antibody 1: 3, anti-B antibody 1: 2, anti-AB antibody 1: 4, anti- D antibody 1: 4, anti-RBC antibody 1: 3. All other antibody solutions are not prediluted but adjusted to 10% (v / v) with methanol.
• the membranes are dried after dispensing the antibodies for 20 min at 40 ° C and then stored at constant humidity until the test.
• a 19 ⁇ 20 mm absorption pad (Whatman Schleicher & Schull, 300) which overlaps the membrane by 3 mm is glued on.
• FIG. 2 and 3 illustrate the design of a lateral flow device suitable for carrying out the detection of a mixed field reaction.
• FIG. 3 shows suitable dimensions which, however, are to be understood as exemplary.
• Diluent F Medion Diagnostics
• Test batch of erythrocyte sediment 50 ⁇ l of erythrocyte sediment (anticoagulated whole blood is centrifuged for 5 min at 1500 rpm, the erythrocyte sediment is in the lower, red-colored phase) are mixed with 400 ⁇ l Diluent F. 100 ⁇ l of the resulting suspension are applied to the application zone. If the application zone is "dry”, apply 300 ⁇ l of Diluent F. The result can be read after about 5 minutes and is expressed by a slightly red wedge ("crescent" - minore cell population) to a strongly red band or strongly red colored point (majore cell population).
• FIGS. 4A and 4B illustrate the detection of a mixed-field reaction with the aid of the two-indicator zone method according to the invention.
• FIG. 4A shows the result of the examination of a blood sample with 100% Acc D.ee and 0% B CCD. EE.
• Figure 4B shows the result of examining a blood sample with 98% A ccD.ee and 2% B CCD.EE.
• FIG. 4B shows the detection of the minimal cell population in the form of a light wedge (crescent) in relation to the major cell population.
• Example 2 Homologous transfusion (blood doping) The test strips were prepared analogously to Example 1.
• the result can be read off after approx. 5 minutes and is expressed by a slightly red wedge ("crescent" - minore cell population) up to a strongly red band or strongly red colored dot (majore cell population).
• test strips were prepared as in Example 1, with only anti-D being applied.
• individual antibodies and not antibody mixtures are applied.
• Preferred antibodies RUM-I (Millipore); LDM-3 (Alba Bioscience); but also ESDIM (Alba Bioscience); TH-28; MS-201; MS-26 (Millipore); LDM-I (Alba Bioscience).
• the result can be read off after approx. 15 minutes and is expressed by a slightly red wedge (minore cell population) up to a strongly red band (majore cell population).
• Example 4 Detection of an erythrocyte characteristic present in extremely low antigenic density: DEL
• test strips were prepared as in Example 3. Preferably, individual antibodies and no mixtures are applied. Preferred antibodies: RUM-I (Millipore); LDM-3 (Alba Bioscience).
• Test batch a 400 ⁇ l of anticoagulated whole blood or freshly collected native blood are mixed with 100 ⁇ l of Diluent F. From the resulting suspension 300 ul are applied to the task zone. If the application zone is "dry”, apply 400 ⁇ L of Diluent F.
• the result can be read off after approx. 20 minutes and is expressed by a slightly red wedge (minore cell population) up to a strongly red band (majore cell population).
• test strips were prepared as in Example 1, with at least 2 (preferably 5) aliquots of the same antibody (anti-RBC) being applied in series.
• Variant 1 The antibody concentrations decrease from proximal to distal;
• Variant 2 There are several traces of flow, each with 2 (5) antibody spots, whereby the antibody concentration decreases from flow track to flow track;
• Variant 3 combination of variant 1 and variant 2;
• Variant 4 The proximal antibody plot is a line followed by several points:
• the result can be read after about 5 minutes (see Figure 5).
• erythrocyte sediment 50 ⁇ l of erythrocyte sediment are mixed with 400 ⁇ l Diluent F. 100 ⁇ l of the resulting suspension are applied to the application zone. If the application zone is "dry", 300 ⁇ l of Diluent F are applied.
• the result can be read after about 5 minutes (see Figure 5).

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