DE2025718B2 - Method for stabilizing lyophilized erythrocytes - Google Patents

Description

This process is a further development of the main patent 18 08 435.

According to German patent 18 08 435, erythrocytes for direct and passive hemagglutination examinations using methylglyoxal and formaldehyde stabilized. Before or after coating with antigen, the erythrocytes are potentiated to the hemagglutination titer to increase.

The object of the invention is to improve the stability of erythrocytes processed with aldehyde, see above that they are particularly suitable for lyophilization. The application therefore relates to a method for Stabilize erythrocytes by treating native erythrocytes separately with both methylglyoxal as well as with formaldehyde in any order according to German Patent 18 08 435, characterized in that that the aqueous erythrocyte preparation suitable for lyophilization, containing 0.1 to 10 % By volume of aldehyde-stabilized erythrocytes 0.2 to about 1% by weight of blood serum albumin and an additional 5 up to 30% by weight of a sugar from the group consisting of sucrose and lactose and mixtures thereof to be added.

Erythrocytes stabilized according to this method show increased activity and greatly improved Storage stability on. Furthermore, these erythrocytes are suitable for a test preparation for indirect or passive hemagglutination study with very superior storage stability.

Native erythrocytes and erythrocytes treated according to already known methods which are suitable for direct Hemagglutination tests, e.g. blood grouping, are useful, have the disadvantage that they spoil quickly in storage, so that they are unusable for many purposes after about 21 days are. In addition, native cells are only suitable for frozen storage in special media and are after lyophilization and reprocessing unsatisfactory. Native red blood cells are also sensitive with regard to the ionic composition, the pH value and the osmotic pressure of the suspension media, so there are undesirable limitations on the scope of tests that can be performed with them can be done.

Erythrocytes coated with known antigens are in approved preparations for the Use as a test agent for antibodies that are specific with respect to such antigens. if an antiserum or a serum containing the suspected antibody with the red blood cells coated is brought into contact, agglutination or clustering occurs, indicating that the suspected Antibody is actually present in the serum. It is already known that such agglutination is a semi-quantitative information regarding the presence and occurrence of specific antibodies and also provides useful information as to the conditions necessary for the presence of such Antibodies give rise to.

There are many problems in the manufacture, storage and use of such coated red blood cells occurred, some of which generally related to the stability of erythrocytes under storage conditions, with their sensitivity in selected passive hemagglutination tests and with their reproducibility related to such hemagglutination tests. The individual erythrocyte cells may agglutinate or show nonspecific hemagglutination, that is, activity that interferes with the hemagglutination study for a specific factor. Coated red blood cells are usually stored in the frozen state, but can be used when thawing frozen preparations Clustering occur or the cells can be destroyed by hemolysis. The keeping of frozen cells in liquid nitrogen or dry ice is expensive and has a considerable impact Space requirement. Transport is particularly difficult. In addition, there is a tendency towards one certain deterioration of the cells on prolonged storage.

On microscopic examination of the cells, such a deterioration occurs through a change the size, shape and evenness. The deteriorated cells form when they work in the hemagglutination examination procedures Use, no sharply defined and tight "button" in the recess and easily cause the hemagglutination titer varies unpredictably from batch to batch of the lyophilized product.

By improving lyophilization of the stabilized erythrocytes, problems such as those above specified, avoided.

Thus, a stabilized erythrocyte preparation is provided, which is particularly suitable for lyophilization suitable is. Furthermore, erythrocyte preparations produced in this way are not after they have been made available for infusion lyophilized, stabilized erythrocyte preparations equivalent.

The erythrocyte preparation provided according to the invention can be stored for longer periods of time without resulting in any hemolysis or agglomeration of blood cells. It can also can be used in hemagglutination tests.

The erythrocyte preparation produced by the method according to the invention can, as said, be effective are coated with different antigens, whereby the coated erythrocytes represent a preparation, that effectively packaged and for longer periods of time until a special passive hemagglutination test is supplied, can be stored.

The invention is described in detail below.

Native red blood cells are found in whole blood from various animals, such as rabbits. Rats. Pigeons.

Sheep and chickens, as well as from humans, collected. The blood sample is isotonic with conventional Treated anticoagulant solutions, then the erythrocytes are compressed, for example by centrifugation, and washed with compatible buffer solutions with an essentially neutral pH, to prevent hemolysis of the erythrocytes and remove unwanted substances that are on the Erythrocytes from the serum may be present.

A suspension of the washed erythrocytes is in the neutral buffer solution in a lesser Concentration of preferably below 10% vol / vol produced. The erythrocytes appear with a smaller one Amount, based on the volume of erythrocytes, of methylglyoxal treated for a period of time that sufficient to give the erythrocytes the desired stability, for example overnight Mix. Following the treatment step with methylglyoxa! erythrocytes become several times washed to remove excess methylglyoxal and any other substances from the serum can originate. The erythrocytes treated with methylglyoxal are then reduced to a smaller amount, based on the volume of erythrocytes exposed to formaldehyde, and the excess formaldehyde is removed by multiple washes with a buffered solution.

The above treatments with methylglyoxal and formaldehyde can be combined as »double Aldehyde treatment ”in order to obtain a“ stabilized erythrocyte preparation ”, see DT-PS 18 08 435. It is essential in the present embodiment according to German patent 18 08 435 that the Treatment with methylglyoxal takes place prior to treatment with formaldehyde, as it has been found that in the case of erythrocytes of certain individuals, an initial formaldehyde treatment leads to damage such cells, which is why this order is recommended. With erythrocytes from other individuals however, an initial formaldehyde treatment may not appear particularly harmful, which is why For some procedures, an initial formaldehyde treatment will still produce beneficial results can lead.

As already mentioned, erythrocyte preparations stabilized according to the invention can be used for considerable periods of time can be stored without undesirable hemolysis, agglomeration or other harmful effects Appearances occur.

Human erythrocytes can be removed by methylglyoxal / formaldehyde treatment can be stabilized without any visible change in their ability to use antisera for blood typing to react. Stabilized human cells of type A, B or AB are specifically or anti-B serum agglutinated. The sensitivity of the agglutination reaction using the stabilized cells, the sensitivity when using native cells is the same or a multiple higher. Type 0 stabilized cells do not react with anti-A or anti-B serum. The above reactivities are maintained for several months when the cells are stored at 4 ° C or in freezer storage. These stabilized cell preparations can be used as reference cells for determining blood groups.

The above stabilized erythrocyte preparation can be converted into a coated stable erythrocyte preparation by contacting a sample of the aldehyde-treated red blood cells with an antigen and then carried out one or more washing steps to remove excess antigen will. In general, the stabilized erythrocytes are coated with antigens in a solution, which is buffered to a pH below 7 and desirably a'if a pH of about 3.6-6. With Polysaccharide antigens, such as E. coli endotoxin, coating can be carried out at pH 7. It results

ίο get superior sensitivity when treated Red cells are conditioned by placing them in the buffered solution before coating with antigen be moved for about 1 hour. The overdrawing step is usually complete in about 1 hour, however

some stoving operations may require 24 hours. An overdraft temperature of 24 ° C is preferred temperatures of 5O ⁰ C or higher provide Erythrozytpräparate with reduced sensitivity. These above conditions can apply to

> o different antigens vary, the skilled person can however, easily the necessary overdrawing conditions in consideration of the occurrence of agglutination determine with serial dilutions of a selected antiserum. For example, a

> ■> Rabbits are injected with various amounts of antigen identical to the antigen adsorbed on the red blood cell specimen. It becomes a serum sample in which the antibodies are present, and serial dilutions of this serum can be stored in plastic

jo shells with a V-bottom are attached to them the same volume of the stable, aldehyde-treated erythrocyte preparation is added. A reaction mixture, which contains cells and diluent, serves as a comparison, in which the cells at the bottom one

π Form a "button", which is a negative reaction. The appearance of a "matted surface" represents a positive agglutination reaction, which indicates satisfactory coating. Non-specific agglutination must be carefully distinguished.

When speaking of an "antigen" coating in describing the present invention is meant to denote a material which may, if not, be an antibody or an antigen especially for special antigens or antibodies

4-Ί is pointed out.

To representative antigens that were successfully used to coat the stabilized erythrocyte preparations Include protein antigens, such as bovine serum albumin or BSA, insolubilized

"> o BSA, human serum albumin, rabbit y-globulin, Human γ-globulin, streptococcal M protein, ragweed plant antigen "E" (ragweed antigen "E"), polysaccharide antigens such as E. coli endotoxin (ET), acetylated ET, succinylated ET, de-esterified ET, de-esterified

-) -, tes and bromoacetylated ET, synthetic polyglucose, mixed antigens such as RNA bacteriophage (ribonuoleic acid), QS, Entamoeba histolytica antigen and other. It is a particular advantage of the present invention that the stabilized erythrocyte preparation for

bii covering with such a variety of Substances with different chemical compositions is suitable.

The hemagglutination titer of either the stabilized or the coated erythrocyte preparation can be

iv ") are increased in that the temperature of the Preparation lowered to about -190 ° C and then at a temperature several degrees below the freezing temperature, preferably at one

uniformly maintained temperature value and at about -20 ° C for a period of about 16 Weeks. On the other hand, there is an equally effective potentiation within of about 6 weeks by changing the temperature of the ■ -, stabilized or coated erythrocytes periodically for several hours and without movement, preferably at intervals of about 6 or 7 days 24 ° C is increased.

Erythrocytes can also be removed by treatment with a dilute solution of an oxidizing agent potentiated, sodium periodate being a suitable reagent.

A potentiated, stabilized red blood cell preparation can be produced by removing its cells before the Coating with the antigen can be coated with fibrinogen. Bovine fibrinogen is a suitable potentiating agent.

Will see to these process measures. Also the remarks in DT-PS 18 08 435. The potentiated, ₂ <> stabilized or coated Erythrozytpräparat may be in a lyophilized state or at a temperature of about -19O ⁰ C stored until it is required for hemagglutination. Improved results in the preparation for infusion are achieved if the cells are lyophilized in an aqueous sucrose or lactose solution which contains a small amount of blood serum albumin. The amount of cells in the solution is not critical and can range from about '/ 10% up to about 10% or more of _M total volume of the preparation may vary. Excellent results are obtained at a concentration between 0.5% and 5%; the preferred concentration is 1%.

Erythrocytes prepared for infusion of the same size, moderately high quality is obtained if the sugar concentration before lyophilization is more than 5% by weight ranges up to about 30% by weight of the solution. Good results are achieved in a concentration range from about 10% to about 20%, and the preferred range is from about 15% to about 20%. the Using sugar at a concentration of less than 5% nonetheless provides some Improvement over a composition in which sugar is absent. Sugar concentrations 4-, above about 30% are less desirable because an inordinate amount of time is required to remove the erythrocytes ready for infusion after lyophilization.

Blood serum albumin from various animals can be used; that of rabbits, chickens and cattle w leads to good results. At least about 0.1% by weight is used to provide the desired To maintain properties in the red blood cells prepared for infusion. Better results will be achieved when the composition contains about 0.3% and a preferred composition contains 0.5% Blood serum albumin. Up to 1% can be used, however a smaller amount will usually be preferred because the blood serum albumin is relatively expensive. Included in the present invention Also the lyophilized erythrocyte preparations, their composition according to the composition the suspension from which they originate varies. The sugar and blood serum albumin are on a weight basis are present and suffer practically no loss during lyophilization. The erythrocyte fraction, which is on a volume basis is present as an aqueous suspension with a density approximating one. loses about 85% their weight when lyophilized. A representative erythrocyte suspension, which consists of 0.1% Cells, 0.2% blood serum albumin and 5% lactose, after lyophilization consists of about 0.3% by weight cells, 3.8 Wt% blood serum albumin and 96 wt% lactose. Similarly, an erythrocyte suspension contains the consists of 10% cells, 1% blood serum albumin and 5% sucrose, after lyophilizing about 20% Cells, 13.3% blood serum albumin and 66.5% sucrose. Similarly, suspensions of 10% yield cells and 1% blood serum albumin in 30% sugar solution and from 0.1% cells and 0.2% blood serum albumin in 30% sugar solution after lyophilization, 4.6% cells, 3.1% blood serum albumin and 92.3% sugar or 0.05% cells, 0.7% blood serum albumin and 99% sugar.

The person skilled in the art is familiar with the manner in which hemagglutination tests are carried out. In general becomes an animal serum sample with the suspected antibody with the uncoated or coated Erythrocyte pooled in a liquid vehicle to determine if agglutination occurs. The stabilized erythrocyte preparations can also be coated with a specific antibody, so that in the subsequent hemagglutination examination the erythrocytes in the presence of a free agglutinate specific antigen. Other Variations and Modifications of the Hemagglutination Testing Procedure are obvious to one skilled in the art, and all such tests can be beneficial with the improved stable and coated red blood cell preparations.

The passive hemagglutination test can be used to determine the presence of γ-globulin determine the cells, as in the Coomb test, for the detection of bacterial infection, as in the presence of endotoxin antigens, for the detection of virus infections, for the determination of tissue tolerance, for the discovery of autoimmunity diseases and other phenomena of this type. the Coated and stable red blood cell preparations can also be used as a test reagent in chemical analyzes to determine the presence of antigen in a quantitative or semi-quantitative manner.

The following examples are presented to illustrate the various embodiments of the invention demonstrate. Although these embodiments include the best methods currently available for practical use Carrying out the invention are contemplated, however, it is to be understood that they are not exclusive Represent embodiments. They also do not necessarily include those embodiments which may be preferred in the future.

example 1

Making a stable one with aldehyde
treated erythrocyte preparation

A 20 ml sample of whole blood is drawn from a 2 to 3 kg rabbit by cardiac puncture. and the sample is collected in a syringe containing an equal volume of sterile Alsever solution contains. This solution is generally isotonic and contains a sodium citrate anticoagulant, sodium chloride, Dextrose and distilled water. The withdrawn sample is left to stand at about 4 ° C. overnight and then centrifuged at 1500 rpm for 10 minutes, whereby about 10 ml of compressed rabbit blood cells

can be obtained. The compressed erythrocytes are then extracted with 10 volumes of a 0.11 molar Phosphate buffer solution with pH 7.2 combined. This buffered solution is made from monopotassium phosphate and Disodium phosphate made by vortex mixing. The mixture of erythrocyte and buffer solution is centrifuged and then 5 separate washes with 10 volumes of the above buffered Solution subjected to remove serum protein and any other serum substances. The washed ones Erythrocytes are then resuspended in the above-mentioned buffered solution to make a rabbit erythrocyte suspension available with 8% vol / vol.

In a 500 ml Erlenmeyer flask, 125 ml of the phosphate-buffered solution, which is 3% vol / vol Methylglyoxal contains, and 125 ml of the above 8% erythrocyte suspension introduced and the The mixture is stirred for about 18 hours at room temperature. The mixture is then poured through a gauze pad filtered to remove any large pieces, and the red blood cells are then removed on 5 separate occasions washed with 10 times its volume in phosphate buffered solution. That with Methylglyoxal treated erythrocyte preparation is then again in the above-mentioned buffered solution suspended at a concentration of 10% vol / vol.

125 ml of the above suspension treated with methylglyoxal is diluted to 8% and mixed with 125 ml of the phosphate-buffered solution containing 3% vol / vol formaldehyde mixed. The mixture is stirred overnight with a magnetic stirrer at room temperature and then through a gauze pad filtered to remove any possible fragments. The ones with methylglyoxal and formaldehyde treated erythrocytes are then on 5 different occasions with ten times their volume washed with the above buffered solution. The red cell preparation treated with aldehyde is then resuspended in the above buffered solution at a concentration of 10% and resuspended in Glass container transferred and sealed for storage. The above aldehyde treated erythrocyte preparation is for at least 2 months at reduced temperatures of 4 ° C or for unlimited periods of time stable when frozen in liquid nitrogen.

The stabilized red blood cell preparation can be potentized, however, in the preferred method the potentization step is only carried out after the erythrocytes have been coated.

The stabilized erythrocyte preparation produced by the method according to the invention has the most, if not all, of the connecting sites present on native red blood cells, with connecting sites specifically refer to the sites that react with an antibody or virus can.

Example 2

Potentiation of a stabilized
Erythrocyte specimen

This example illustrates the increase in herding agglutinal ion titre achieved by Fibrinogen with the cells of the stabilized erythrocyte preparation connected. Bovine fibrinogen is used at a concentration of 1 microgram per milliliter in 0.1 molar acetate-buffered solution with pH 4.0 hour at 60 ° C and then heated to R & umtempe cooled down. A 10% suspension of the double aldehyde treated Erythro

-> cells obtained by following the procedure of example 1! is added to the fibrinogenesis solution and win Stirred for 1 hour at room temperature. The potentiated stabilized erythrocytes are mi a phosphate buffer solution with pH 7.2 washed around

κι the erythrocyte concentration is set to 10% vol / Vo set. These cells are later used for blood typing with excellent results used.

Example 3
Coating a potentized stabilized

Erythrocyte specimen

1 ml of the 10% suspension of the potentized stabilized erythrocyte preparation, which according to the The method of Example 2 is obtained using a

r> 0.1 molar acetate buffer solution with pH 4.0 washed and resuspended in 9 ml of the acetate buffer solution at 24 ° C. 1 ml of the acetate buffer solution, the 1 mj Containing bovine serum albumin is added. The mixture is lanj for 2 hours at room temperature

stirred in and then 5 times with an excess of mi Phosphate buffered solution with pH 7.2 washed these cells later for passive hemagglutina tion reactions used.

Example 4

Covering a stabilized
Erythrocyte specimen with antigen

Be 24 ° C given 1 ml of the 10% suspension of the aldehyde <treated erythrocyte preparation, which according to dei Process steps of Example 1 is obtained. The SI

4i sample is then diluted with a 0.1 molar acetate buffer solution solution washed with pH 4.0. The stabilized erythrocytes are not at this lowered pH value hemolyzes and does not clump together. The washed erythrocyte preparation cells are in 9 m

■] () of the acetate-buffered solution with pH 4.0 at 24 ° C resuspended and it is stirred at this pH and at this temperature for 1 hour. Then we( 1 ml of the acetate buffer solution containing 1 mg of bovine serum albumin was added. The mixture of antigen and <

v> stable erythrocytes treated with aldehyde will be Rotated room temperature for 2 hours followed by 5 separate washes with excess Volumes subjected to a phosphate-buffered solution with pH 7.2. The one with beef serum albumir

w) coated stable erythrocytes are in the Resuspend the above phosphate buffer in a concentrator of 0.5% vol / vol and it will 10 ml aliquots of this suspension are packed in 20 m glass vials and sealed. The packed!

hi predetermined amount of coated stable mi Aldehyde-treated erythrocyte preparation is then razed in a refrigerator with liquid nitrogen frozen and stored at -20 ° C.

Example 5 Potentiation of the coated, stable erythrocyte preparation

Treated erythrocytes as in Example 4, but at a pH value of 3.6, coated and - Frozen 196 ° C and 6 days at -2O ⁰ C have been stored long, without moving to room temperature of 24 ° C thawed, kept for 2 hours at 24 ° C and brought back to -20 ⁰ C after refreezing at -196 ⁰ C. The procedure is repeated once a week for 6 weeks.

The potentiated, coated erythrocytes are then frozen at -196 ° C and at this temperature stored until needed for the hemagglutination test. Alternatively, the suspension of the potentiated, coated cells and lyophilized by adding water before use for infusion be prepared.

This procedure increases the titer 30-fold, with the non-specific hemagglutination titer only is doubled, which is a practically insignificant increase. Further repeated freeze-thaw cycles lead to a further increase in the hemagglutination titer, but also call a simultaneous one significant increase in the non-specific haemagglutination titer, so that from the Extended processing has little advantage.

The optimal number of freeze-thaw cycles is related to the special coated cell preparation; the person skilled in the art knows, however, that the optimum lies in the cycle beyond which the increase of the hemagglutination titer from a noticeable increase in the non-specific hemagglutination titer is accompanied. A coated cell preparation that is optimal for the standard serum is also for others Antisera was found to be optimal. Thus, successive standard sera can be used against the originally chosen antiserum to be standardized.

Example 6 Potentiation of a stabilized erythrocyte preparation

This example illustrates another embodiment of the potentiating agent of the present invention Invention. Human erythrocytes are processed according to the procedure of Example 1 to obtain stabilized Human erythrocytes suspended in a phosphate-buffered solution (pH 7.2). The stabilized Human erythrocytes are then soaked in 0.00035 molar sodium periodate solution at 24 ° C for 15 minutes treated and then washed free of this reagent using a phosphate buffered solution (pH 7.2).

As the stabilized cells of the direct hemagglutination reaction to rat antiserum gave it at a dilution of 1: 100

in a reaction, while the stabilized cells potentiated by the oxidation reaction contribute to hemagglutination at a dilution of 1: 2000.

The following example illustrates the lyophilization of red blood cells to provide for

π Infusion prepared cells of high quality.

Comparison attempt

A solution of stabilized erythrocytes coated with bovine serum albumin (BSA) and prepared according to the Procedure of Example 4 were prepared, was at a concentration of 1% in piner 0.11 molar, with phosphate-buffered solution with sucrose and rabbit serum albumin suspended in such a way that in the final solution gave a concentration of 15% and 0.5%, respectively. The solution was following standard working practices lyophilized and stored in the lyophilized state for several days. The lyophilized Erythrocytes were then made up for infusion with water and a hemagglutination reaction used against anti-BSA serum # 8. The results are presented as Experiment b, in Table I along with the Results of a comparison, designated as experiment a, are shown with those with non-lyophilized cells was carried out.

Solutions were also made with various other concentrations of sucrose and lactose for that Lyophilization prepared according to the procedure of the comparative experiment. The results of the hemagglutination reactions with the erythrocytes prepared for infusion «. 'are grounded in Table 1 as Experiments c to k shown. Note that cells treated with 0.5% rabbit serum albumin and 20% lactose had been lyophilized, in its mode of action, from cells that had not been lyophilized were indistinguishable. The replacement of lactose with sucrose in an amount of about ■ in 15 to about 20% gave similar results. at Slightly lower concentrations of sugar and rabbit serum albumin was an increase in the Hemagglutination titers observed; the mode of action was nevertheless excellent.

4i The cells lyophilized according to this procedure can be prepared for infusion and lyophilized repeatedly without adverse effects.

The desirable properties found in the lyophilized and prepared for infusion, lyophilized Cells are detected, result solely from the use of the blood serum albumin in Combination with lactose or sucrose. A preparation which was similar to that of experiment c, but did not contain blood serum albumin, called a hammock

v, agglutination titer of I 280,000 and control "buttons" of poor quality. In the same way unsatisfactory results were recorded in experiments in which a similar preparation as that of experiment c, but no sucrose or

containing lactose was used.

A preparation containing 10% sucrose and 4% normal rabbit serum gave a hemagglutination titer of 1,280,000 and control "buttons" of poor quality. It is therefore clear that the

hi presence of sucrose or lactose together with blood serum albumin is critical to the successful practice of the present invention.

Table 1

Lyophilized, stabilized, bovine serum albumin coated cells; Hemagglutination titer against anti-BSA serum number 8.

example Suspending media containing Hemagglutination Quality of 0.11 molar phosphate buffer plus titer, reciprocal Control "buttons" a non-lyophilized 160,000 excellent b 15% sucrose-0.5% RSA *) 160,000 excellent C. 20% sucrose-0.5% BSA 160,000 excellent d 20% lactose-0.5% BSA 160,000 excellent e 10% sucrose-0.5% BSA 320,000 excellent f 20% sucrose-0.2% BSA 320,000 excellent G 10% lactose-0.5% BSA 320,000 excellent H 15% lactose-0.5% BSA 320,000 excellent i 5% lactose-0.5% BSA 640,000 excellent k 10% sucrose-0.1% BSA 640,000 excellent

*) RSA = rabbit serum albumin.

Claims (1)

Claim: Process for stabilizing erythrocytes by treating native erythrocytes separately both with methylglyoxal and with formaldehyde in any order according to German patent 1808435, characterized in that the aqueous erythrocyte preparation suitable for lyophilization, containing 0.1 in to 10 vol / o of aldehyde-stabilized erythrocytes, 0.2 to about 1% by weight of blood serum albumin and additionally 5 to 30% by weight of a sugar from the group of sucrose and lactose and mixtures thereof are added. ι ^r >