CH646793A5 - In vitro method for diagnosis of malignant tumors. - Google Patents

Description

646 793

PATENT CLAIM In vitro methods for diagnosing malignant tumors, preferably in humans, which method determines the change in mobility of leukocytes that come into contact with a migration inhibition factor, called MIF, that is secreted by lymphocytes that are sensitive to tumor tissue are, the change in mobility being measured in a migration inhibition test, in the course of which the lymphocytes, as part of a leukocyte suspension, are brought into contact with an antigen solution which stimulates the formation of MIF and are then incubated, and after a fixed incubation period, the Leukocyte migration is inhibited and fixed, characterized in that the sample contains an antigen solution according to Caspary and Field or a 3 m KCl extract from tumor tissue according to Meitzer in the ratio - volume sample to the antigen solution - from 1:50 to 1:10 and in addition a transfer factor solution is added.

The invention relates to an in vitro method for the diagnosis of malignant tumors, preferably in humans, in which test the mobility change of leukocytes is determined which comes into contact with a migration inhibition factor, called MIF, which is excreted by lymphocytes which are sensitized to tumor tissue, the change in mobility being measured in a migration inhibition test, in the course of which the lymphocytes, as part of a leukocyte suspension, are brought into contact with and then incubated with an antigen solution which stimulates the formation of MIF, and after a fixed incubation period, leukocyte migration is inhibited and fixed.

It is known [Ax, W. and Tautz, C., Behring Inst. Mitt. 54 (1974), pages 72 to 80] to carry out a migration inhibition test under agarose, with which a migration inhibition of the leukocytes of cancer patients is detected has been. However, no reproducible results were obtained when using soluble tumor antigens (tumor extracts).

It is therefore the task of improving the known migration inhibition test so that it can also be used to examine tumor-antigen solutions.

This object is achieved in a method of the type mentioned at the outset, in which the sample consisting of the leukocyte suspension, an antigen solution according to Caspary and Field or a 3 m KCl extract from tumor tissue according to Meitzer in the ratio (volume sample to Antigen solution) 1:50 to 1:10 and in addition a transfer factor solution is added.

A test is carried out on a sample with leukocyte-rich supernatant ("buffy coat") obtained from human blood with a fixed number of leukocytes between 104 and 10 'per sample. These standard values are tried and tested values that do not require excessive blood sampling from the patient.

An antigen solution in a ratio of 1:50 to 1:10 is added to the buffycoat sample. Antigen solutions can be used which are prepared by a method specified by Caspary and Field (Caspary, EA et al., Brit. Med. 1971, pp. 613 to 617, and other publications; see Douwes, FR et al., Verh German Ges. Inn. Med. 82, JF Bergmann-Verlag, Munich 1976) or with the aid of a 3 m KCl extraction according to Meitzer et al. [Meitzer, M.S. et al., J. Nat. Cancer Inst. 47 (1971) p. 703 ff.]. Human malignant tissue is used as the starting material

Operations or human brain matter. However, it is also possible to obtain antigen solution from tumor cells grown in vitro.

The method according to the invention can not only be applied to an unspecific malignancy examination, but can also be extended to an organ-specific examination. If antigens are used which have been obtained from carcinoma tissue of certain organs, lymphocytes from patients affected by the same organ carcinomas react in the manner described. The literature describes that e.g. antigen solution obtained from mammary carcinoma according to Caspary and Field or Meitzer result in a specific reaction exclusively for lymphocytes which originate from patients with mammary carcinoma.

It is also possible to have a positive immune response in a general test, i.e. if a carcinoma is suspected, carry out a series of tests with organ-specific antigens and thus find out in a relatively short time which organs could be affected by a carcinoma.

It is very important that the so-called immune response, i.e. the elicitation of the slowing-down effect during the movement of the indicator cells in the present tests can surprisingly be made reproducible by adding a solution with a so-called transfer factor to the sample solution. This factor is known from immunology [Schröder, I. et al., Z. Immun.-Forsch. 149 (1975), pp. 365 to 371], the transfer factor can change non-reactive lymphocytes in such a way that they release mediators of cellular immunity after incubation with the relevant antigen. However, it could not be expected that when using transfer factor with antigen solutions, reproducible results, such as with culture cells as antigen, can be obtained. It is therefore surprising that an in vitro test method for the diagnosis of malignant tumors is possible if an additional transfer factor factor solution is added to the antigen solution.

The transfer factor can be made from normal blood leukocytes. It represents a subcellular, dialysable, non-immunogenic leukocyte factor, which is responsible for the T-lymphocyte-dependent reaction. Transfer factor preparations are available (Schura, Krefeld), which are produced by fractionation in liquid oxygen and subsequent gradual ultrafractionation. One commercially available unit contains a transfer factor of approximately 5 x 1010 leukocytes. The preparation is stabilized with 10% human albumin solution.

It is shown that an increase in the immune response can be achieved by adding a transfer factor. The addition of the transfer factor therefore significantly improves the discrimination against the individual trial tests.

The method is conveniently carried out with a test plate made of a flat carrier plate or shell made of inert material, e.g. Glass, on which a 2 to 5 mm thick solidified agar layer is applied, with a capillary migration layer of 1 to 20 μm thick being between the carrier plate or dish and the agar layer and the agar layer two essentially has circular punch holes with a diameter between 0.5 and 5 mm, preferably 2.5 mm. Each punch hole has a capacity of about 5 to 20, preferably 8, ul of test substance.

It is advantageous if the punched holes are cut so that the uniformity of the capillary migration layer is also given in the region of the transition from the punched hole to the layer.

The invention is illustrated by means of examples. Fer

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The following figures are attached:

Fig. 1 shows a test plate for performing the method according to the invention in plan view;

Fig. 2 shows a test plate in section.

example 1

A. Obtaining the leukocyte suspension

30 ml of human blood is heparinized with 300 USP units of Na heparinate. The heparinized human blood (patient's blood) is sub-layered in a plastic tube with 8 ml of 6% by weight dextran solution in physiological saline (molecular weight of the dextran: 75000). The erythrocytes are then allowed to sediment at 37 ° C. in a warming cabinet for about 45 minutes. The leukocyte-rich supernatant (buffy coat) is siphoned off and mixed with the same volume of 0.9% by weight NaCl solution, then centrifuged for 15 min with an acceleration of 750 g. The pellet which separates out in the centrifuge is again washed twice in 10 ml Hank's solution (pH 7.2) and suspended in a Tc 199 medium (manufacturer: Serva, Heidelberg). When washing and suspending, the cells are whirled up with a pipette until no macroscopic clumps can be seen. After staining with Turkish solution, the leukocytes are counted in the Neubauer chamber and adjusted to 2-105 cells / ul in Tc 199 medium. The mean results for the sample are 25% mononuclear cells (lymphocytes and monocytes) and 75% granulocytes. Erythrocyte contamination is less than 1 to 1.

B. Preparation of the antigen solution

To produce the antigen solution, the encephalito-gene factor (EF) or the basic protein from carcinoma tissue (CaBP) must be isolated.

The tumor or brain is processed under sterile conditions as soon as possible after the surgical removal. Connective tissue and healthy tissue are removed from the tumor. The tumor or brain tissue is then cut into small pieces using sterile scissors (scalpel) and further crushed in a homogenizer in an ice bath. The tumor tissue must not warm up during this process. The homogenate is suspended in four times the volume of distilled water and then centrifuged at 23,000 g. The resulting pellet is resuspended in four times the volume of 0.9% NaCl solution, homogenized and centrifuged at 23,000 g for 30 min. The pellet, again resuspended by four times the volume of distilled water, is freeze-dried. The freeze-dried powder is mixed 1:10 with a chloroform / methanol mixture (2: 1) and stirred for 30 min. The mixture is centrifuged at 600 g for 10 minutes. The supernatant is discarded.

This process is repeated twice. The pellet is air dried. The dry pellet is resuspended in five times the volume of a 5% NaCl solution in water and centrifuged at 23,000 g for 30 min. The supernatant is discarded. With the addition of n / 100 NaCl, the pellet, again resuspended in five times the volume of distilled water, is adjusted to a pH of 3.5. The suspension is shaken gently for 30 min, then centrifuged again at 23,000 g for 30 min. The supernatant is discarded. The pellet is resuspended again in five times the amount of distilled water, which is adjusted to a pH of 2.6 with n / 100 HCl and left to stand for 3 to 18 hours. The pH value must not change during this time. The suspension is centrifuged at 23,000 g for 30 minutes. The supernatant is saved, the pellet is washed again in n / 100 NaCl at pH 2.6 and centrifuged. The resulting supernatant is saved. The latter two supernatants are brought together and then dialyzed against distilled water. This is followed by freeze drying.

An antigen test solution is obtained by dissolving 0.1 mg / ml of the freeze-dried substance in distilled water. The manufacturing process, which is basically the same, also applies to a solution of the basic protein from tumor tissue (CaBP).

C. Production of the test plate (cf. also FIGS. 1 and 2)

0.5.g of a special culture agar (agarose; manufacturer Behring-Werke, Marburg) is weighed into 50 ml Erlenmeyer flasks. 22.5 ml of sterile aqua bidestillata are added.

22.5 ml of the double-concentrated Tc 199 medium and 5 ml of plasma obtained from human blood are placed in a second Erlenmeyer flask. The agarose is dissolved in a holder in a boiling water bath while gently shaking in a circular motion. The Erlenmeyer flask is closed with a foil or a stopper, otherwise part of the water would evaporate.

After the agarose has completely dissolved, it is allowed to cool to 47 ° C. in a water bath. The medium-plasma mixture is then preheated in the same bath and added while shaking the agarose, so that 50 ml of an 1% agarose are now present in simple Tc 199 medium with 10% plasma.

A preheated pipette is used to fill 5 ml of the agar into round tissue culture dishes (Petri dishes) 10 with a diameter of 50 mm, which are shown in plan view in FIG. 1. After the agar has solidified, holes 11 with a diameter of 2.5 mm are punched into the agar using a telescopic punching cannula (Behring-Werke, Marburg) with the aid of a template. When punching out, make sure that the agar layer in the punching area is not detached from the glass layer, so that the capillary layer does not enlarge and so the migration of the leukocytes is no longer guaranteed.

The arrangement of the holes 11 results from FIG. I. Of course, further figurations are conceivable. FIG. 2 shows an enlarged section through the lower part of the dish 10. It is indicated that the agar layer 1 is separated from the glass layer of the dish 10 by a thin capillary layer 3. The individual rows of holes are intended for example for:

A control (standard)

B Test suspension with general antigen

C Test suspension with organ-specific antigen

D Test suspension with organ-specific antigen and transfer factor.

D. Reaction, incubation and fixation

The following approaches were made or mixed for a test:

1. 100 jil leukocyte suspension + 8 (il antigen solution.

2. 100 μl leukocyte suspension + 8 ml antigen solution + 20 u.1 solution transfer factor (preparation Schura; 1 u.1 corresponds approximately to the transfer factor of 5 x 105 leukocytes).

3. 100 ml of leukocyte suspension.

4. 100 ul leukocyte suspension + 20 | i.l transfer factor solution as in approach 2.

The mixed batches 1 to 4 are incubated for 60 min at 37 ° C. in a warming cabinet at 100% atmospheric humidity.

Then 8 and 1 leukocyte- and antigen-containing substance or standard substance are filled into each punch hole from the incubated solution. For the evaluation of leukocyte mi5

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Depending on the configuration of the test plate (number of punch holes), 2-5 measurement results and 2-5 reference values are available, depending on the configuration of the test plate (number of punch holes) and the influence of the factors produced by the lymphocytes, which slow migration. The cells contained in the suspension, which are inserted into the punch holes at the start of the experiment, migrate into the intermediate layer 3 between the agar and the surface of the dish. By incubating the plates at 37 ° C in the warming cabinet but for 18 hours, this migration is maintained over a defined period. The leukocytes do not migrate into the agar, but only get the nutrients from it. Incubation is carried out in a humidity cabinet at a relative humidity of 100%.

After the incubation, the plates are covered with methanol for 15 minutes and then with formalin solution (37% by weight of methanal) for 10 minutes. The agar is then carefully pulled off the skin as a skin. The cells that have migrated in the intermediate layer are now fixed on the surface of the shell. After drying, the cells are stained according to Pappenheim, resulting in dark purple halos 4 (migration yards) on the plate surface (cf. FIG. 1).

E. Measure and determine the results

The diameter of the migration halo is measured with a magnifying glass (Bausch & Lomb). The polymorphonuclear cells lying in the periphery must also be recorded. The arithmetic mean is formed from several measurements.

The quotient of the migration area Fi with antigen and the migration area F2 without antigen results in a “migration index” MI:

MI = Ft / F 2-

A migration index <0.85 means significant inhibition of leukocytes and thus a positive cell response. The following table shows a measurement example:

line

0 (mm)

F (mm2)

MI

B

6.75

Fi = 35.8

0.80

A

7.55

Fi = 44.75

(1)

Example 2

Use of an organ-specific antigen.

A. The leukocyte suspension is obtained as in Example 1A.

B. Preparation of an organ-specific antigen solution

If possible, freshly obtained tumor tissue from breast cancer is freed of blood, adipose tissue and connective tissue and cut into small cubes or strips. All subsequent steps are carried out at 4 ° C. The tissue fragments are cut into approximately pea-sized pieces in a triple volume of a 3 molar potassium chloride solution. The pH of this coarse suspension is adjusted to 7.4. The tissue is then crushed using an Ultra-turrax® homogenizer (20,000 rpm). The homogenate is left to stand for 24 hours. It is then centrifuged. The supernatant obtained after centrifugation at 4000 g for 60 minutes is dialyzed for 2 hours against a 10-fold volume of aqua destillata and then for a further 24 hours against 50-fold volume of 0.9% NaCl solution. The dialysate is added by adding 2 molar ammonium sulfate

The proteins precipitated for a period of 1 hour and were separated off by centrifugation at 4000 g for 20 minutes. After suspension of the proteins in a 0.9% NaCl solution, dialysis is carried out for one hour against 5 times the volume of aqua destillata and another dialysis against 50 times the volume of 0.9% NaCl. After sterile filtration through a 0.45 Milipore Micropore filter, the antigen extract is filled into small ampoules and stored at - 20 ° C. Before use, the protein content of each batch is determined using the biuret method after previous TCA precipitation.

C. The same test plate as in Example 1 is used.

Three approaches are made for the test:

1. with 100 u.1 leukocyte suspension from patient blood according to Example 1A + 8, ul n / 100 NaCl solution (for row A, standard value);

2. with 100 | il leukocyte suspension + 8 and 1 antigensus pension according to Example 1B,

3. with 100 ul leukocyte suspension + 8 u.1 antigen suspension according to Example 1B + 15 ixl transfer factor solution.

The test plate with a hole diameter of 2.5 mm is charged with 8 ul of the test substance 1-3 incubated according to Example 1. The results, each determined from five values, are:

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0

F (mm2)

MI

1

A

7.00

46.5

1.00

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7.70

38.1

0.82

3rd

D

6.10

29.3

0.63

The result indicates breast cancer, since the MI is well below 0.8 and the result is even more pronounced when the transfer factor is used.

Breast cancer patients using the method with the aid of the transfer factor show a migration index of over 90%, which is less than 0.80. The migration index is greater than 0.85 in only 10% of breast cancer patients.

Example 3

The change in mobility of lymphocytes, foreign macrophages and other indicator cells is measured in a capillary test according to Soborg and Bendixen. A 10 µm thick open capillary is used. The results correspond to those of Example 1, the same approaches being used in principle.

Closing remarks

The test procedure is not applicable for certain clinical pictures. False negative results were found for all so-called leukoses. An explanation for this cannot be given at present, since leukemic cells in particular contain very abundant basic proteins. The cellular immunity in these patients is not so noticeably disturbed that this result can be explained by a disturbance in the immune system. It is therefore also striking that chronic lymphatic leukemia, like acute lymphatic leukemia, does not show any reaction, while lymphosarcomas and lymphogranulomatoses behave like the other malignancies. Before chemotherapy, however, patients had a significant inhibition. However, this inhibition is removed after the first chemotherapy cycle and converted into a false positive value in the second cycle. Furthermore, there are incorrect results in patients with tumor cachexia. Patients with

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Rapidly progressively growing tumors or patients in tumor cachexia sometimes show incorrect results because there is a disturbance in cellular immunity. Attempts to answer this question are still ongoing.

False positive results were found primarily in inflammatory and degenerative neurological diseases, for example multiple sclerosis. The explanation for this was seen by Caspary and Field in a cross reaction between the EF and the sensitization of the lymphocytes with brain tissue.

However, the wrong results can easily be eliminated by a preliminary examination, so that the diseases mentioned do not prevent the test from being used.

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1 sheet of drawings