DE2755363C3 - Test method for diagnosing malignancies - Google Patents

Description

The invention relates to an in vitro method for diagnosing malignant tumors, preferably in People, in which test the change in mobility of leukocytes is determined, which with a Migration Inhibition Factor (MIF), which is secreted by lymphocytes, which are sensitized to tumor tissue, the change in mobility in a migration inhibition test is measured, in the course of which the lymphocytes as part of a leukocyte suspension with a for Formation of MIF stimulating antigen solution brought into contact and then incubated, and after a fixed incubation period, the migration of leukocytes is inhibited and fixed. -

It is known. (Ax, W. and Tautz, C, Behring Inst. Mitt. 54; 1974; pp. 72-80), a migration inhibition test to be carried out under agarose, which inhibits the migration of leukocytes in cancer patients has been proven. However, there were none when using soluble tumor antigens (tumor extracts) to obtain reproducible results.

The task is therefore to improve the known migration inhibition test to the effect that that also tumor antigen solutions can be examined with it are.

This object is achieved in a method of the type mentioned at the outset, in which the sample consists from the leukocyte suspension, an antigen solution according to CASPARY and FIELD or a 3-m-KCl extract from tumor tissue according to MELTZER in a ratio (volume sample to antigen solution) of 1:50 to 1:10 and a transfer factor solution is also added.

A test is carried out with a sample with leukocyte-rich supernatant ("buffycoat") obtained from human blood with a fixed number of leukocytes between 10 ⁴ and 10 ⁷ per sample. These standard values are proven values that do not require excessive blood withdrawal from the patient.

An antigen solution in a ratio of 1:50 to 1:10 is added to the buffycoat sample. Here you can Antigen solutions are used which are prepared according to a method specified by CASPARY and FIELD (Caspary, E A. et aL, Brit Med. 1971, pp. 613-617 and more publishments; see Douwes, F. R. et al .; Proprietary German Ges. Inn. Med, Volume 82, J. F. Bergmann-Verlag, Munich 1976) or with the aid of a 3 m KCl extraction according to MELTZER et al. (MELTZER, M.S. et al, J. Nat Cancer Inst. 47; 1971; P. 703 ff.). Serve as starting material

ίο human malignant tissue from surgery or human brain matter. However, it is also possible to use antigen solution from tumor cells grown in vitro to win.

The test method according to the invention can not only be applied to a non-specific malignancy examination » but also expand to an organ-specific examination. Are used antigens that come from Carcinoma tissue of certain organs have been obtained, this is how lymphocytes react from patients who are attacked by the same organ carcinomas, in the manner described in the literature described that z. B. obtained from breast cancer according to CASPARY and FIELD or MELTZER Antigen solution will result in a specific response exclusively for lymphocytes from those sick with Breast cancer originate.

It is also possible, if there is a positive immune response, in a general test, i.e. in Carcinoma suspected, 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. H. causing the slowing effect in the Movement of the indicator cells in the present tests, surprisingly made reproducible as a result can be that the sample solution is added a solution with a so-called transfer factor. This Factor is known from immunology (Schröder, 1. et al., Z. Immun.-Forsch., 149; 1975; pp. 365-371). Of the Transfer factor can change non-reactive lymphocytes in such a way that they are affected after incubation with the Release antigen mediators of cellular immunity. However, it could not be expected that when using transfer factor with antigen solutions reproducible results as with culture cells are available as antigen. It is therefore surprising that an in vitro test method for the diagnosis of Malignant tumors become possible if a transfer factor solution is added to the antigen solution adds.

The transfer factor can be produced from normal blood leukocytes. It represents a subcellular, dialyzable, non-immunogenic leukocyte factor

which is responsible for the T-lymphocyte-dependent response. Transfer factor preparations are available (SCHURA, Krefeld), which are produced by fractionation in liquid oxygen and subsequent step-by-step ultra-fractionation. A commercially available unit receives a transfer factor of approx. 5 · 10 ¹⁰ leukocytes. The preparation is stabilized with 1% human albumin solution.

It turns out that the addition of transfer factor enhances the immune response.

The addition of the transfer factor therefore significantly improves the discrimination between the individual test tests.
The procedure is carried out with a test plate

leads from a flat support plate or tray inert material (e.g. glass) to which a 2 to 5 mm thick, solidified agar layer is applied, being between the support plate or tray and the agar layer is a capillary migration layer from 1 to 20μηι thickness and the agar layer at least two substantially circular punched 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 μΐ test substance.

It is advantageous if the punched holes are cut in such a way that the uniformity of the capillary migration layer is also maintained in the area of the transition from the punched hole is given to the shift

The invention is illustrated by means of examples. The following figures are also attached:

F i g. 1 shows a test plate for carrying out the method according to the invention in plan view; F i g. 2 shows a test plate in section

Example 1 A Obtaining the leukocyte suspension

30 ml of human blood are heparinized with 300 USP units of sodium heparinate. The heparinized human blood (patient blood) is placed in a plastic tube with 8 ml of 6% by weight dextran solution in physiological saline solution (molecular weight of dextran: 75,000) Sediment erythrocytes at 37 ° C in an oven for about 45 minutes. The leukocyte-rich supernatant (buifycoat) is siphoned off and mixed with the same volume of 0.9% strength by weight NaCl solution, then centrifuged for 15 min at an acceleration of 750 g. The pellet which separates out in the centrifuge is washed two more times in 10 ml each time of Hank's solution (pH 7.2) and suspended in a Tc 199 medium (manufacturer: Serva, Heidelberg). The cells are washed and suspended with a pipette whirled up until macroscopically no more cell clumps can be seen. After staining with Türk's solution, the leukocytes are counted in the Neubauerkammer and ^{adjusted to 2 · 10 5} cells / μΐ in Tc 199 medium. On average, the sample yields 25% mononuclear cells (lymphocytes and monocytes) and 75% granulocytes. The erythrocyte contamination is less than 1 to 1.

B Preparation of the antigen solution

The encephalitogenic factor (EF) or the basic protein is used to produce the antigen solution Isolate carcinoma tissue (CaBP).

The tumor or the brain will be under sterile conditions as soon as possible after the Surgical removal processed connective tissue and healthy tissue are removed from the tumor. Thereafter the tumor or brain tissue is cut into small pieces with sterile scissors (scalpel) and im Homogenizer further comminuted in an ice bath. The tumor tissue must not be removed during this process heat. The homogenate is suspended in four times the volume of distilled water and then centrifuged at 23,000 g. The resulting Peilet is again in four times the volume of 0.9% NaCl solution suspended, homogenized and centrifuged for 30 min at 23,000 g That again in four times the volume distilled water resuspended pellet is freeze-dried The freeze-dried powder is im Ratio 1:10 with a chloroform / methanol mixture (2: 1) are added and the mixture is stirred for 30 minutes. The mixture is centrifuged at 600 g for 10 minutes. The upper class is discarded.

This operation is repeated twice. The pellet is air-dried. The dry pellet is im Resuspended five times the volume of a 5% NaCl solution in water and centrifuged for 30 min at 23,000 g The upper class is discarded. With the addition of n / 100 NaCl this is again five times as high Volume of distilled water resuspended pellet adjusted to a pH of 3.5 The suspension is 30 min shaken gently then centrifuged again for 30 min

ι5 23,000 g. The supernatant is discarded. The pellet is again resuspended 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 min The supernatant is retained, the pellet is washed again in n / 100 NaCl at pH 2.6, centrifuged, the resulting supernatant is removed here. The latter two

Supernatants are pooled and then dialyzed against distilled water. Then takes place a freeze drying.

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

C Preparation of the test plate
(See also Figs. 1 and 2)

0.5 g of a special culture agar (agarose; manufacturer Behring-Werke, Marburg) are placed in 50 ml Erlenmeyer flasks weighed in. To this end, 22.5 ml of sterile double-distilled water are added.

22.5 ml of the double concentrated Tc 199 medium and 5 ml Given plasma obtained from human blood. The agarose is placed in a holder while gently rotating Shake dissolved in a boiling water bath. The Erlenmeyer flask is covered with a foil or a Stopper closed, 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 to the agarose while shaking, so that 50 ml of a 1% agarose in simple Tc 199 medium with 10% plasma are now present.
A preheated discharge pipette is used to fill each 5 ml of the agar into round tissue culture dishes (Petri dishes) 10 with a diameter of 50 mm, which are shown in FIG. 1 are shown in plan view. After the agar has solidified, holes 11 with a diameter of 2.5 mm are punched into the agar with a telescopic punch cannula (Behring-Werke, Marburg) with the aid of a template so that the capillary layer does not enlarge and so the migration of the leukocytes is no longer guaranteed.

The arrangement of the holes It is shown in FIG. 1. Further figurations are of course possible conceivable. The F i g. 2 shows an enlarged section

- wmpm ~ -it ± tcJl

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 is separated by a thin capillary layer 3. The individual rows of holes are for example reserved 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 are used or mixed for a test:

1. IOP μΐ leukocyte suspension + 8 μΐ antigen solution

2. 100 μΐ leukocyte suspension + 8 μΐ antigen solution

+ 20 μΙ solution transfer factor (SCHURA preparation; 1 μί corresponds approximately to the transfer factor of 5 · 10 ⁵ leukocytes)

3. 100 μΐ leukocyte suspension

4. 100 μΐ leukocyte suspension + 20 μΐ transfer factor solution as with approach 2.

The lugs 1 to 4 are for 60 min at 37 ° C in a heating cabinet at 100% humidity incubated Then μΐ of the incubated solution to each punched hole S leukocyte and antigen-containing substance and the standard substance filled For the evaluation of leukocyte migration and the influence of the Factors that slow down migration produced by the lymphocytes are available, depending on the configuration of the test plate (number of punched holes), 2–5 measurement results and 2–5 reference values. The cells contained in the suspension, which were introduced into the punch holes at the beginning of the experiment, migrate into the intermediate layer 3 between the agar and the surface of the dish. This migration is maintained over a defined period by incubating the plates at 37 ° C in a heating cabinet for 18 hours. The leukocytes do not migrate into the agar, but only draw the nutrients from it. The incubation is carried out at a relative humidity of 90-100% in a humidity cabinet

After incubation, the plates for 15 minutes with methanol and then 10 min with formalin (37 wt .-% methanal) overcoated Subsequently, the agar is carefully removed from the skin as a tray bottom. The cells that have migrated in the intermediate layer are now fixed to the surface of the dish. After drying, the cells are colored according to Pappenheim, producing dark purple halos 4 (wandering halos) on the surface of the plate (see Fig. 1).

E measuring and determining the results

The diameter of the wall halo is measured with a measuring magnifier (Bausch & Lomb). The isolated lying in the peripheral polymorphonuclear cells are to detect with. The arithmetic mean is calculated from several measurements

The quotient of the migration area Fi with antigen and the migration area F ₂ without antigen results in a "migration index" MI:

WED - F, / F ₂ .

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

line

0 (mm)

F (mm ² )

6.75
7.55

F ₁ = 35.8 F ₂ = 44.75

A shell with four by five punched holes (2.5 mm diameter) is produced. One row each with 5 holes is charged with 10 μΐ of the test batches 1 to 4. Results (each averaged from five values):

approach line 0 (mm) F (mm2) Ml 1 B. 6.90 37.4 0.79 2 C. 6.05 28.7 0.61 (incl. Transfer factor) 3 A. 7.75 47.2 1.0 standard 4th D. 7.70 46.6 0.99

The low MI of rows B / C indicates a case of malignancy.

Results with the migration inhibition test below Use of the transfer factor in the determination of gastrointestinal tumors:

Measurements were carried out using the measurement procedure according to Example 1. In a (healthy) patient control group there was no significant inhibition, the migration index was 1.02 ± 0.1. The transfer factor did not influence these results. In the group of patients suffering from colon carcinoma, the Migration index at 0.65 ± 0.2. Of the 15 cases examined, 11 showed a migration index, which was less than 0.85. After adding transfer factor, the migration index was 0.61 ± 0.09. About that In addition, in 14 of 15 patients the MI after the addition of transfer factor was below 0.85, so that here, too, one significant improvement in the test result had been achieved. In other groups studied no significant inhibition could be found.

Example 2 Use of an organ-specific antigen

A The leukocyte suspension is obtained

as in Example 1-A
B Creation of an organ-specific antigenic nourishment

Breast cancer tumor tissue obtained as freshly as possible is freed from blood, adipose tissue and connective tissue and cut into small cubes or strips. The following steps are carried out at 4 ° C. The pieces of tissue are crushed into pea-sized pieces in a three-fold volume of a 3m potassium chloride solution. The pH of this coarse suspension is adjusted to 7.4.

Homogenizer (20,000 rpm) comminuted. The homogenate is left to stand for 24 hours. It is then centrifuged. The supernatant obtained after 60 minutes of centrifugation at 4000 g is dialyzed for 2 hours against a 10-fold volume of aqua distillata and then for a further 24 hours against a 50-fold volume of 0.9% NaCl solution. The proteins are precipitated from the dialysate by adding 2M ammonium sulfate over a period of 1 hour and separated by centrifugation at 4000 g for 20 minutes. After suspending 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-Miüpore micropore Fiker the antigen extract is filled in. Small vials and stored at -20 ⁰ C, the protein content of each batch is determined with the biuret after previous TCA precipitation before use

C It will be the same test plate as for Example 1 used

Three approaches are made for the test:

1. each with 100 μΐ leukocyte suspension from patient's blood according to Example 1-A + 8 μΐ "/ ₁₀₀ NaCl solution (for row A, standard value)

2. each with 100 μl leukocyte suspension + 8μ1 Antigen suspension according to Example 1-B,

3. each with 100 μΐ leukocyte suspension -I- 8 μΐ Antigen suspension according to Example 1-B + 15 μl transfer factor solution.

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

approach

line

F (mm *)

MI

A. C. D.

7.70 7.00 6.10

46.5 38.1 29.3

1.0

0.82

0.63

The result suggests breast cancer as the MI is well below 0.85 and the result is at Application of the transfer factor is increased.

Patients with breast cancer show when using the method with the aid of the

10

15th

20th

25th

30th

40

45

50 Transfer factor at over 90% a migration index that is less than 0.80. Only in 10% of the patients with Breast cancer, the migration index is greater than 0.85.

Example 3

It is the change in mobility of lymphocytes, foreign macrophages and other indicator cells in a capillary test according to S0BORG and BENDIXEN. It is here a 10 μm thick open Capillary used. The results correspond to those of Example 1, but in principle the same approaches be used.

closing remarks

The test procedure cannot be used for certain clinical pictures. Gave false negative results with all so-called leukosis. An explanation for this cannot be given at the moment, as it is leukemic cells contain very abundant basic proteins. Cellular immunity is also with these Patients are not so noticeably disturbed that this result could be explained by a disturbance of the immune system. It is therefore also noticeable that chronic lymphatic leukemia, just like acute lymphatic leukemia Leukemia show no response, while the lymphosarcomas and lymphogranulomatoses behave like the other malignancies. Before chemotherapy, however, patients showed significant inhibition on. After the first chemotherapy cycle, however, this inhibition is lifted and im second cycle converted into a false positive value. Furthermore, incorrect results are obtained in patients with tumor cachexia. Patients with rapidly progressive tumors or patients in the Tumor cachexia often show incorrect results, since there is a disruption of cellular immunity Attempts to answer this question are still ongoing.

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

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

For this purpose i sheet of drawings

Claims (1)

Claim: " In vitro method for the diagnosis of malignant disease Tumors, preferably in humans, in which test the change in mobility of leukocytes which come into contact with a migration inhibition factor (MIF) determined by Lymphocytes are excreted, which are sensitized to tumor tissue, with the change in mobility is measured in a migration inhibition test, in the course of which the lymphocytes as part of a leukocyte suspension with an antigen solution that stimulates the formation of MIF in Brought into contact and then incubated, and after a fixed incubation period the leukocyte migration is inhibited and fixed, characterized in that the The sample contains an antigen solution according to CASPARY and FIELD or a 3 m KCl extract from tumor tissue according to MELTZER in a ratio (volume sample to antigen solution) of 1:50 to 1:10 and additionally a transfer factor solution is added.