DK153425B - PROCEDURE FOR DETERMINING CELL TYPE OR COMPATIBILITY ON A FIXED MATRIX. - Google Patents

Description

DK 153425 B

The present invention relates to a method for determining cell type or compatibility on a solid matrix.

For many medical purposes, it is necessary to determine the compatibility of blood from a donor with a patient.

Blood cell compatibility is determined by the lack of an immunological response between antibodies contained in a patient's blood serum and antigens present on blood cells from a donor. For example, if a patient's red blood cells are of type A (i.e., have "A" antigens on the red blood cells), the serum from such patient's blood will contain anti-B antibodies, ie. antibodies that will react with "B" cells. If such a patient receives a transfusion of "B" blood, an immunological reaction will occur between the anti-B antibodies in the patient's serum and the B antibodies from the donor's red blood cells. Such incompatibility

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can result in serious consequences due to intravascular hemolysis.

Blood cell type and compatibility studies usually consist of two types: (i) a study to determine if a specific antibody upon addition to the blood cells will effect their agglutination, and (ii) a study to determine whether a specific antibody by Addition to the examined blood cells together with serum complement will cause blood cell lysis.

The first of these two basic studies, the agglutination, refers to a clumping of blood cells containing e.g. type A antigens to which anti-A antibodies are added in the absence of complement. The A-antigen and anti-A antibodies specifically react with each other in an immunological reaction in which the antibody forms bridges between adjacent cells. This leads to a closely linked mass of blood cells which are joined to each other by the added antibodies.

The second of the two studies discussed above, cell lysis, concerns the breaking of cell membranes leading to cell death and release of their intracellular contents. "Cell lysis" is the result of a reaction that occurs between cell membrane-bound antibody and a group of potentially destructive proteins in normal serum (often called "complement").

Both methods described above are used for typing and compatibility testing of the cellular blood elements, erythrocytes, granulocytes, lymphocytes and platelets (thrombocytes). Although often used only qualitatively, both methods can actually be quantified, and have been used separately to determine antigen, antibody, and serum complement.

In the blood cell typing and compatibility testing methods commonly used in the clinical field today, both agglutination studies and cell lysis studies are performed in a liquid phase, i.e. that sera containing antibodies with or without complement to be examined is mixed with suspensions of the blood cells for which blood type or compatibility is to be assessed. Usually, fixed volumes are used.

Assessment of agglutination assay results requires the technician to distinguish agglutination of cells due to specific antigen-antibody molecular bridging from non-specific cell aggregation, at which extraneous forces also cause

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of clumping. Thus, the technician must be able to distinguish free unagglutinated cells that may be present from clumped or agglutinated cells. This requires highly experienced staff or precise particle sorting and counting with expensive instruments. In addition, measuring the degree of specific agglutination is either poor enough semi-quantitative or costly and complicated to complete.

Although some instrumented studies have been developed to characterize red blood cells by agglutination, the equipment for these methods is both costly and complicated to use. P.eks. is one device that has been proposed for the typing of red blood cells by instrumental, known as "AutoAna lyzer" and manufactured by Berkman et al., described in Transfusion, Vol. 11, No. 6, pp. 317, et seq. . (1971). In said equipment, blood samples and antibody sera are combined under special circumstances in complex tubular spirals designed to effect agglutination. The reaction coils then pass a "T" piece with the bone in a downward position so that the agglutinates that are formed tend to be removed. Agglutination can be detected by measuring the decrease in optical density in the discharge stream of the "T" which results in the non-agglutinated fraction (Berkman et al.), Or by trapping the agglutinates from the "T" on filter paper (Shield et al. , Transfusion, Vol. 9, pp. 348, 1969). However, the apparatus is complex and costly and cannot be used for a simple automatic technique suitable for routine use in blood bank examinations.

An alternative device is known as "Groupamatic" and can cost several hundred thousand dollars (see Garretta et al., Vox Sang., Vol. 27, pp. 141, 1974). In the Groupamatic equipment, sera and blood cell suspensions are combined to produce agglutination. The occurrence of agglutination is demonstrated by passing the suspension over two rays of light, one passing through the center of the reaction cuvette while the other passing through the periphery. A difference in the transmission of the jets is taken as the measure of the agglutination strength.

However, sophisticated electronic equipment is required which brings the instrument out of reach of anyone but the largest blood banks.

Immunolysis-based studies present no problem when the cell surface antigen-antibody reaction provides effective reciprocal interaction with complement as for tissue (e.g., HL-A) typing. Unfortunately, this is rarely the case with red human blood cells, where either the cell membrane antigen-antibody complex cooperates.

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may be ineffective with complement or the antibody concentration must be limited to prevent intense agglutination, which will mechanically interfere with the immune light.

These problems seriously affect the operation of blood bank serology laboratories, where even routine red blood cell typing becomes a time-consuming manual operation that requires more skilled and experienced staff than can be obtained. In addition, relatively few blood banks may undertake lymphocytotoxic studies necessary for tissue typification. There are no direct immunological studies available to determine pre-transfusion compatibility. the mobility of granulocytes and only an indirect study for platelets such as ^ C serotonin release. (Although cm granulocytes and platelets may be assigned to HL-A types by selected and appropriate studies, such typing will not guarantee their compatibility).

It has now surprisingly been found that both agglutination and cell lysis studies on human blood cells can be significantly improved when a reactive cell monolayer is irreversibly bound to a solid matrix.

Thus, the present invention provides a method for determining cell type or compatibility on a solid matrix which is characterized by: (a) forming a first monolayer of cells such as erythrocytes that irreversibly bind to a solid matrix which has inherent or (b) contacting the layer (a) with a solution containing antibodies which can be bound by immunoadsorption to the first layer (a) of cells to the extent that the antibodies in the solution are reactive with the antigens in the cells of the first layer, (c) then applying a suspension of other cells, such as erythrocytes, which other cells have antigens thereon, and (d) measuring the degree of immunoadherence of the layer of other cells that bind with antibody to the first cell layer.

By irreversible binding of cells to a solid matrix is meant binding of reactive cells by molecular forces, such as formation of covalent chemical bonds between positions on the cell surface and reactive groups on the substrate, or formation of bonds at weaker molecular forces, such as van der Waals forces, columbaric or hydrogen bonding which will withstand the effect of the further process conditions. Not only is the sensitivity of

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the investigative method increased, but the results of the immune response are easy to measure using simple instruments.

As a simple illustration, said blood typing study can be performed in three steps: (1) a monolayer of erythrocytes (for illustration, type A will be referred to) irreversibly bound to a solid matrix; (2) using the cell monolayer as immunoadsorbant, antibodies such as anti-A antibodies can be specifically applied and adsorbed; and (3) the antibody-coated cells can now be examined for their ability to bind another monolayer of cells carrying appropriate antigens (solid phase agglutination).

The examination result can be evaluated in any convenient way, such as by examination under a microscope; of particular importance to the present invention, however, is that the study results are particularly suitable to be assessed by densitometric techniques using readily available standard instruments.

For solid-phase hemagglutination, the test plate prepared in the foregoing manner may be subjected to static or scanning densitometry at wavelengths where the hemoglobin content of the investigated erythrocytes is light-absorbing (e.g., blue light having a wavelength of 415 nm is suitable). Scanning densitometry involves only the use of well-established laboratory equipment that is readily available and provides quantitative answers to questions such as (1) Is a second layer formed? (2) How many cells does that second layer represent? and ØD How is the distribution of the second layer? (by "distribution" is meant the uniformity of the second layer. A uniform second layer implies that 100% of the cells in the applied suspension carry the necessary antigen for binding to the first layer, whereas a non-uniform distribution implies the presence of some " negative "cells in the applied suspension).

In the above described method for assessing immunological factors on cell surfaces, the presence of antibodies or antigens immunoadsorbed from the solution can be conveniently detected by applying a different suspension of the cell population to be examined after the bound monolayer has been reacted with antigen. (or antibody) solution, and measuring the formation of another cell layer wherein the antigen (or antibody) with

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known specificity acts as a crosslinking agent.

The process of the invention may also be used to investigate competing reactions between solutions or suspensions of materials suspected of sharing antigenic properties. Thus, an irreversibly bound monolayer of cells is prepared as described above. This layer is then contacted with a mixture of two solutions, one containing antigens or antibodies of known specificity capable of being immunoadsorbed on the bound cell monolayer, and another solution or suspension to be investigated. Competitive binding of the known antibodies or antigens with factors present in the second solution or suspension will be reflected by a reduction in immunoadsorption reaction formation with the irreversibly bound cell monolayer.

Parallel to this method is the preparation of a cell monolayer with a second layer of antigens attached thereto as described above. Competitive reactions can then be measured between a suspension of cells capable of forming another cell monolayer and another solution or suspension containing unknown factors to be analyzed. The occurrence of competing immunological reactions between these two suspensions (or suspension and solution) will be reflected by a reduction in the formation of another cell monolayer.

Within these general guidelines, the following specific assay methods may be envisaged: 1. Analysis of antigens by competing immunoadsorption. This analysis comprises steps of (a) applying to a solid matrix a first suspension of cells known to carry antigens to be examined under conditions effective to irreversibly bind a layer of the cells to the matrix; (b) contacting layer (a) with a mixture of (i) a solution of an antibody reactive by immunoadsorption with antigen carried by the cells of layer (a), and (ii) another solution or suspension which must be assayed for antigen by competing inhibition of the antibody in solution (i); and (c) then measures the degree to which the antibody in solution (i) is bound by immunoadsorption to layer (a).

2. Analysis of antigens by competitive inhibition. This analysis comprises steps of (a) applying to a solid matrix a first suspension of cells known to carry antigens to be examined under conditions effective to irreversibly bind a layer of the cells to the matrix; (b) applying to layer (a) a solution containing an antibody reactive by immunoassay.

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7 node adsorption with antigens carried by the cells of layer (a); and (c) then applying a mixture of (i) another suspension of cells of the population and (ii) a suspension or solution containing an antigen that is reactive with the immunoadsorbed antibody from solution (b) and measures the degree to which cells from the second suspension form a second layer of cells on the matrix.

3. Analysis for antigens in the presence of antibodies in a cell population. This analysis comprises steps of (a) applying to a solid matrix a first suspension of cells known to carry antibodies under conditions effective to irreversibly bind a layer of the cells to the matrix; (b) contacting layer (a) with a mixture of (i) a solution to be assayed for antigen; and (ii) another solution or suspension containing antibodies reactive by immunoadsorption with the antigen to be assayed; by competing inhibition; and (c) then measures the degree to which the antigen in solution (i) is bound by immunoadsorption to layer (a).

In each of the foregoing methods, the degree of immunoadsorption can be determined by one or more of the techniques generally described above.

Although the clinical problems associated with blood bank serology have persisted for a long time, and despite the need to improve the methods and make these methods suitable for instrumentation and automation techniques, there has been little help for those skilled in the art in the art. the prior art. The so-called "Eldon" blood typing cards have been known for a number of years. These cards are e.g. disclosed in U.S.A. No. 2,770,572, which discloses a test card for use in the typing of human blood, where samples of test sera containing antibody factors in admixture with conglutinin or conglutinin substitutes are placed on different parts of the surface of a carrier sheet.

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When conducting blood typing studies using the Eldon card, blood samples from the patient whose blood is to be typed are placed in small droplets on the various serum spots found on the card and examined after an appropriate reaction time has elapsed, for the presence or absence of agglutination. . However, the studies are limited to anti-A, anti-B and anti-Rh (RhQ or D), and even these studies are associated with significant interpretive errors1.

Less recently, in the U.S.A. U.S. Patent No. 3,666,421 discloses another diagnostic test medium in which serological reagents are placed in droplets on a slide and dried to a spot which can later be reconstituted and converted into an agglutination reaction to identify blood type (or other antigen-antibody reaction systems which can be identified by agglutination). However, these sample glasses suffer from the deficiencies characteristic of ordinary liquid-phase agglutination, the study showing essentially only the presence or absence of agglutination, and the determination of whether the agglutination results from specific antigen-antibody reaction or simply the result of a non-specific cell aggregation depends on the assessment of a qualified technician.

It is difficult or impossible to assess whether free non-agglutinated cells are present with clumped cells in specific agglutinates.

IN USA. U.S. Patent No. 3,770,380 discloses a device which is proposed to be used for the assessment of immunoadherence reactions. In this connection, it should be noted that the immunoadherence reactions to which this patent relates differ from the immunoadsorption phenomenon on which the present invention is based. Immunoadherence is the non-specific clumping of particles or cells due to the presence of complement, and in this case Sample is the complement that causes binding. Immunoadherence is characterized by nonspecific reactions between the complement and the particles and cells to which it binds, in contrast, immunoadsorption is a specific link between antigenic positions on a cell membrane and antibodies present in a serum. Thus, in contrast to immunoadherence, immunoadsorption refers to a specific binding between antigens and antibodies.

The equipment described in this patent is a flat cell-like structure which is provided on the bottom with successive coatings of a bacterial or viral material as a top layer which

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is bound to the cell foundation by means of a transparent dried protein substrate, immunoadherence between bacteria or virus on the unit thus produced and red blood cells bearing antibody and complement, in a test fluid supplied thereto, is then assessed by determining the degree to which the specially pretreated red blood cells in the infused fluid adhere to the bacterial or viral top layer. The method described in the patent has not found practical clinical application because immunoadherence itself has not been widely used and because immunoadherence is a non-specific reaction not suitable for the assessment of specific antigen-antibody reactions.

A layer of blood cells embedded in a solid support material has been used for scientific purposes which are unrelated to blood typing and compatibility testing. Such layers are not bound by covalent or other molecular forces. Goodman (Nature, 193: 350, 1962) thus prepared columns of formalin-treated human red blood cells embedded in polyurethane, which were used to fractionate human red blood antibodies based on the strength of their binding to the retained red blood cells. Edelman et al. (Proc. Nat. Acad. Sci. 68: 2153, 1971) fractionated blood cells based on their capacity to specifically bind to lectins, antibodies or antigens which had been previously bound to partially hydrolyzed nylon fibers.

The principle of binding a prosthetic group (of an antigen, antibody, enzyme, etc.) to a solid matrix is a well-established biochemical method (Cuatrecases and Anfinsen, Annu. Rev. Biochem., 40: 259, 1971), and it is widely used in solid phase radioimmunoassay methods (Brit. Med. Bull. 30: 1-103, 1974). However, blood or other cells have not been irreversibly bound to a solid matrix to facilitate blood or tissue typing, compatibility testing, etc.

The invention is described below in relation to the currently used studies of erythrocytes. Polystyrene test tubes are coated with fibrinogen (0.5 mg / ml used) which irreversibly binds. After leaching, polylysine (0.1 mg / ml) is applied to the bound fibrinogen. Upon further leaching, a suspension of either normal or protease-treated erythrocytes (RBC) is introduced. These RBCs are irreversibly bonded (for study purposes) as a monolayer to the polylysine fibrinogen coated polystyrene surface. The bond density of 2 x 10 RBC per cm is in good agreement with

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ctet, could be expected for such erythrocytes having a diameter of crude 7 µm.

The monolayer of bound RBCs is stable and will in turn serve as an immunoadsorbent for binding antibodies from a solution (or serum) specific for antigenic positions on the membranes of the bound RBCs.

The method of binding blood cells as a monolayer on a solid matrix is not necessarily limited to the above purpose. The literature on coupling proteins to polymers (Cuatrecases, and Anfinsen, Annu. Rev. Bichem., 40: 259, 1971) shows that, as an alternative, polymer preparations (layers of polyurethane, polystyrene, etc.) can be used as on their surface contains reactive groups such as glutaraldehyde, cyanogen bromide, amino or carboxyl groups and others which will allow direct covalent coupling of the blood cells to the matrix. It is obvious that the binding substance must be immunologically inactive for any of the antigens or antibodies that may be present in the study solutions intended to be used.

Other substrates or matrices for use in the invention may be any convenient material which is (i) of such a nature that a cell monolayer can be irreversibly bonded to it as described above, and (ii) suitable for use when the detection method used is taken in consideration. Since the most convenient detection methods are based on light transmission, such as microscopic counting and densitometric scanning, it is preferred to use a light transparent substrate.

However, if radioactivity counts are used, it is obvious that use of material which is transparent to light is unnecessary.

For purposes of the invention, the substrate or matrix may simply be the inner surface of a test tube. If a densitometric scanning method is to be used to assess the study results, a flat surface is appropriate. For large-scale purposes, strips of matrices with cell-binding properties can be used to prepare the first cell layer. Then, antibodies can be stained and examined for their capacity to either support immune lysis in the presence of complement or to bind another layer of cells of unknown type. The quantitative result of both studies can then be determined by scanning densitometry, with loss of color indicating lysis and the addition of color indicating binding of another layer of red cells. For the last purpose, it may be appropriate

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as part of the pie pretreatment hypotonically brighten the first cell layer / so that the background due to the first layer need not be subtracted, Hypotonic lysis does not significantly remove much membrane bound antibody and formation of a second layer can be detected visually or by optical densitometry using its haano-globin content. Using scanning densitometry, e.g. at 415 nm, quantitative answers can be obtained for questions such as "Is a second layer formed?", "How many cells (expressed as an open maximum) does the second layer represent?", "What is the distribution in the second layer?".

To answer the last question, the cell suspension applied to the antibody-coated monolayer should have just enough concentration to allow precipitation of another monolayer.

The second layer is then washed to remove unbound cells, and after this leaching, the distribution of the bound portions of the second layer becomes a function of the percentage of "positive" cells adhering and of "negative" cells which do not hangs on. The holes or islands resulting therefrom can be detected by microscopic scanning densitometry, and their frequency and extent can be expressed as a relative part of the scanned surface.

It should be noted that the binding of antibody by immuno-adsorption to a layer of red blood cells offers numerous significant advantages. First, sera with antibody concentrations that are too low for conventional liquid phase studies can be used successfully because their specific antibody content can be concentrated as bound antibody on the monolayer of cells. Second, under circumstances where undiluted sera cannot be used for liquid-phase studies due to other disruptive serum proteins, such disruption in solid-phase studies by selective adsorption of the antibody to be investigated and leaching to remove the disruptive serum proteins. Third, many sera are useless for liquid-phase studies because they contain antibodies of undesirable specificity that cannot be removed. According to the present invention, by appropriate selection of the cells forming the first layer, it is possible to adsorb only the antibodies to be assayed.

The present invention is suitable for investigating both cell type and cell compatibility. For cell typification e.g. For example, the first layer of its bound antibody may be constructed of cells and antibodies of known type. The type of unknown cells from a patient

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or donor may then be determined by their ability to form another cell layer. For the purpose of conducting compatibility studies, donor cells may be used to form the first monolayer, which is then loaded with possible antibodies in a patient's serum which will bind by immunoadsorption. This can then be demonstrated by determining the ability of the bound antibodies from the patient's serum to form a second layer of the same donor cells after another application (and the degree to which such binding occurs).

Detection methods suitable for use in the present invention will be apparent to those skilled in the art. Erythrocytes contain their own label, namely pigmented protein (hemoglobin), which has a maximum absorption at 415 nm. Therefore, the presence or absence of erythrocytes can be conveniently detected specifically as described above. However, other marking techniques can be used equally well if desired. Such techniques include the use of radioactive atoms (e.g., CrCr, ½), biochemical technique (e.g., a selected intracellular enzyme), or fluorescence detection (e.g., using a molecular sample to identify either a living or dead cell). All these methods can be easily instrumented and therefore subject to automation. They are equally applicable to blood bank serology, forensic blood testing, tissue typing as well as pre-transfusion and pre-transplant examination for compatibility.

As is well known in the art, there are a number of methods now used in liquid phase studies which will be equally suitable for solid phase studies. These include the optimal use of additives known to potentiate agglutination (e.g., symmetric and asymmetric hydrophilic colloids, proteases, ionic concentration, polyelectrolytes, tonicity and buffer systems for pH control). These factors are described e.g. by Berkman et al., in Transfusion, 11: 317, 1971.

It should be noted that the present invention may also be used in conjunction with other problems involving immuno-specific cell responses. One such is antiglobulin assays to evaluate cells for their coating with IgG, IgM, IgA,

IgD and IgE immunoglobulins and with C3, C4 and other complement components or bound activation products (Rosenfield et al., Vox-Sang., 26: 289-333, 1974). Another area of application would be fixed-phase studies with quantitative assessment of both passive and reverse passive hemagglutination studies. Passive hemoglobin

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tination assays can be used for direct analysis of antibody concentration and also for indirect analysis of soluble antigen concentration by competing binding on the basis of common antigens (Nusbacher et al. / J. Immunol. / 108: 893, 1972). Reverse passive studies measure soluble antigen directly (Cook, Immunol., 8:74, 1965; and Juji and Yokochi, Japan, J. Exptl. Med., 39: 615, 1969). Just as blood cells can be examined for their susceptibility to antibody - mediated agglutination or lysis in solid phase studies, further bacteria, protozoa, fungi and cultured cell lines from either tissue or tumors can be assayed for antigenic components on their surface by the solid phase studies described herein. . It is even expected that solid-phase studies can eventually be used to solve problems related to molecular antibody concentration, K value for antibody binding, and degree of K value heterogeneity.

It is recognized that many of the underlying chemical principles used in the present invention are well known. However, the invention is unique in that it has not been previously recognized that these principles could be utilized for the design of solid-phase cell monolayers for effective blood cell typing and blood and other cell compatibility studies.

The invention will now be described in more detail by way of example.

Example 1 ·

Blodtypificering

The problem of typing red human blood cells and detecting Cold human blood antibody antibodies at the point of pre-transfusion compatibility study is considerable. Thus, the only means of detecting vicious blood types of red human blood cells by direct agglutination have been costly and complicated instruments (Berkman, E.M. et al., Transfusion, 11: 317, 1971). However, instrumented liquid-phase methods have been successfully adapted for solid-phase investigation, and specific typing has been achieved for Rh, Kell, Kidd, Duffy, Xg, Lewis, Lutheran and MNSs, all with a sensitivity which exceeds that obtained with the instrumented liquid-phase studies described.

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For blood typing, a monolayer of red blood cells was constructed as follows: using Falcon polystyrene test tubes (10mmID x 75mm) 0.2 ml of fibrinogen solution (0.5 mg / ml) was applied for 2 minutes followed by after leaching, application of 0.2 ml of 140,000 mole weight poly-D-lysine HBr solution (0.1 mg / ml) for 2 minutes. After further leaching, 0.2 ml of 2-5% (v / v) suspension of type A cells was applied in 0.9% NaCl for 2 minutes. This resulted in the adhesion of a flat monolayer of red 6 2 blood cells with a density of 2 x 10 10 cells / cm. These red blood cells remained adherent despite numerous leaches.

The monolayer thus obtained was first exposed to known specific antibody (0.2 ml) and then hypotonic with distilled water. At this point, another application of 0.1 ml of red blood cells in 0.2% (v / v) strength is allowed to precipitate by the effect of gravity as a slightly different monolayer. It was now possible to increase the specific antibody binding of this second monolayer in various ways. Eg. Berkman et al.'s lavion method was successfully used by first replacing the above fluid with a solution of 5% mannitol and 0.0025% protamine sulfate at pH 6.0, and then washing it after 5 minutes at room temperature. second monolayer with 0r0014M phosphate with 0.85% NaCl as buffer with pH 7.3. This removed non-antibody-bound known negative cells but not antibody-bound known positive cells. However, other methods of increasing the antibody binding of the cells were found to be even more advantageous and some of them could not be successfully used in Berkman's instrumented liquid-phase method. Thus, the specific antibody binding of the second monologue could be enhanced by sequential replacement of the above fluid first with a buffer of pH 7.0 containing 2.5% PVP, and then with a similar buffer of pH 6.0 containing 0.01% protamine sulfate. These samples were finally washed with 0.2M phosphate buffer with The pH of 7.3 It is clear that the possibilities of enhancing solid-phase agglutination are speech rings because the method avoids many of the problems associated with liquid-phase studies.

These solid-phase studies have been found to be extraordinarily sensitive to the detection of IgG Rh antibody. Approximately the same sensitivity has been achieved on an antibody molecule per red blood cell basis as described previously for improved AutoAnalyzer assays, i.e.

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10 antibody molecules per blood cell at 50% hemagglutination (Rosenfield et al., Ann. N.Y. Acad, Sc., 190: 519, 1971). In the solid phase studies, however, 1/1000 fewer red blood cells are used, and 1000 times stronger sensitivity is obtained, so that approx. 1 µg antibody protein / ml, which exceeds the sensitivity of all previously described studies, including the in vivo cell survival studies.

Anti-Rh studies were also successfully conducted with protease-treated red blood cells. Such studies were performed on flat bottom microtiter polystyrene dishes which allowed microscopic examination of specifically adhered red blood cells. Bound cells were present only if Rh positive, and in studies of artificial mixtures of Rh positive and Rh negative red blood cells, "holes" of non-adherent cells in area were observed to correspond to the percentage of Rh negative cells. in the artificial mixture. This result shows that, according to the invention, the ratio of unbound cells in a blood sample can be determined quantitatively, which is a particularly significant problem both in analysis of transfusion cell survival by the Ashby method (Arch. Int. Med., 35: 516, 1925). and in characterizing human chimeras (Race and Singer, "Blood Groups in Man", Davis, 1968, pp. 475-490).

Example 2

Direct antiglobulin studies

These studies were conducted similarly to blood typing studies except that washed red blood cells from a patient with suspected acquired hemolytic anemia were used to build both the first and second cell monolayers. The first monolayer (bound with polylysine fibrinogen) was exposed to a single xenogenic anti-human globulin serum and seven specificities were evaluated. These sera were individually specific for IgG, IgM, IgA, IgD, IgE, C3 and C4. The antibody-coated first monolayer was then hypotonically lysed and washed before applying cells to the second monolayer. The binding of the second monolayer was enhanced by the addition of 1% K-90 polyvinylpyrrolidone (PVP, average molecular weight 300,000) in 0.9% NaCl. The second monolayer was finally washed with plain 0.9% NaCl. The procedure is very similar to that of Hsu et al. (Vox Song, 26: 305, 1974), but positive results in solid-phase study were

Claims (1)

16 DK 153425 B clearly outperforms the results of Hsu's instrumented liquid-phase studies. A patient with actively acquired hemolytic anemia who, due to intense spontaneous liquid-phase agglutination with PVP, could not be typified for adherent proteins, was found to be clearly positive for IgG, IgM, IgA, IgE, C3 and C4. A method for determining cell type or compatibility on a solid matrix characterized by (a) forming a first monolayer of cells, such as erythrocytes, that are irreversibly bound to a solid matrix which have in-dwelling or acquired antigens, (b) contacting the layer (a) with a solution containing antibodies which can be bound by immunoadsorption to the first layer (a) of cells to the extent that the antibodies in the solution are reactive with the antigens in the cells of the first layer, (c) then apply a suspension of other cells, such as other erythrocytes, which other cells have antigens thereon, and (d) measure the degree of immunoadherence of the layer of other cells that bind with antibody to the first cell layer.