DD139903A5 - TEST METHOD FOR THE DIAGNOSIS OF MALIGNOMES - Google Patents

Description

Test method of diagnosis of malignant tumors

Field of application of the invention

The invention relates to a test method for the diagnosis of malignant tumors and to a test plate for carrying out the method.

The method according to the invention is used in medicine for the early detection of cancer in humans. In particular, the invention relates to an in vitro assay for the diagnosis of malignant tumors, preferably in humans, in which method the change in the mobility of cellular blood constituents which come into contact with a migration inhibitory factor (IvIIF) is determined, the MIF of Lymphocytes is excreted, which are sensitized to tumor tissue. Such a MIF will not be excreted if the lymphocytes are from patients who do not have a malignant tumor (malignancy), since these lymphocytes are not sensitized, and therefore on contact with. Tumor antigen does not release MIF.

From the literature (Douwes, R. et al., Verh. German Gesum- tern, Medo 82, Band, J _e F Bergmann-Verlag, Munich 1976, there further evidence) is known that with the help of the so-called. Electrophoresis Mobility Test (EMT) is an early detection of malignant diseases possible.

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The principle of the EMT due to the fact that the migration velocity (mobility) visible from the specific, in the electric field of a special microscope (Zytopherometer) bodies (macrophages, granulocytes _s other blood cells) only _s changes when they come in contact with Lymphokineη from sensibilxsierten lymphocytes " It is assumed

that lymphocytes; which are sensitized to a specific antigen (so-called T lymphocytes), release a number of soluble factors, the so-called lymphokines. An important factor among these factors is migration inhibition factor (MIF). It affects the mobility of blood cells, particularly macrophages and granulocytes, and provides for their arrest and accumulation at the site of the presence of the sensitizing antigen. In vivo, this means the possibility of destroying the tumor.

Lymphocytes of malignant patients react specifically with the release of the so-called soluble factors when they come into contact with a basic protein taken from the brain substance on the one hand or from a carcinoma tissue on the other hand. The baseline proteins, which cause the specific reactions of lymphocytes, are called in the literature encephalitogenic factor (EF) and basic protein from carcinoma tissue (Cq ₁ BP). The recovery is carried out according to methods known per se (Caspary, EA et al., Brit Med, 1971, pages 613-617 and other publications, see Douwes, FR aa Ö).

The results of literature described in the literature

Blood tests with the EMT show that with the help of the test, a nearly 100% differentiation of the samples can be made as to whether they have been taken from an organism with malignant (tumor) diseases or a healthy organism or a non-malignant organism. All examined patients with (malignant) tumors showed a positive lymphocyte response, i. H. lymphocytes have given rise to a factor which leads to a measurable and characteristic change in the mobility of indicator cells (see Douwes, F.R., a "a" 0.) Control studies on patients with non-malignant diseases did not show such a lymphocyte response. False results could be attributed to certain causes. Accordingly, the EMT can be considered as currently the most sensitive cancer test.

A disadvantage in the application of the test is felt that the control of a relatively complicated-to-use instrument, namely the cytopherometer, is required. The personnel observing and measuring the rate of migration must be trained and monitored very carefully. Due to the diminishing attention, only a limited amount of work is possible. The application of the EMT is limited

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therefore specialized institutes. The evaluation of the. Tests are carried out during the movement of the blood corpuscles, as'o dynamic »- A" stop "of the experiment is not possible, therefore, no remeasurement is carried out.

Object of the invention

The aim of the invention is to provide a test method for the diagnosis of malignancies, the most possible and unspecific with respect to the type of malignancies with high accuracy signals the presence of malignancies, but principle in each doctor's lab set up and can be performed and especially for mass examinations in the prevention of cancer eigiEb. The result of the test should be easily readable. The test should be statistically evaluable and archived without intermediate logging »

Explanation of the essence of the invention

The invention has for its object to modify the known migration inhibitioris test so that the result of Tesbe is nachmeßbar.

This object is achieved by an in vitro test method for the diagnosis of malignant tumors, in which test the mobility change of cellular blood components t that with a migration inhibition factor

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(MIF) is determined, which is excreted by lymphocytes, which are sensitized to tumor tissue in which as a cell-shaped blood components, the lymphocytes to be examined as part of a leukocyte suspension subjected to a known migration Inhibrbions test in the course of which the lymphocytes are brought into contact with an antigen that stimulates the formation of MIF and then incubated

and after a fixed period of time the Leu is inhibited and fixed.

There are various migration inhibition tests are known, which are basically suitable for the investigation according to the task. Known among other things, the so-called. Capillary technology according to S0BORG and BENDIXEN. To realize the concept of the invention, it is necessary to measure the mobility of the indicator cells by "freezing" a migration state reached after a certain time.

According to the invention, the propagation or mobility change of the LOCACs is preferably measured within a very fine, about 10, thick "open" capillary layer. Particularly suitable, a test arrangement has been found, which consists of a two-layer element, namely from a carrier of an inert medium, for. Glass, and an agar enriched with nutrients for blood cells. Particularly suitable as agar is an agar commercially available under the name Agarose, which is manufactured by Behring-Werke, Marburg.

The intermediate layer between agar and carrier layer

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is preferably partially filled with liquid. It has a thickness of about 1-20 .mu.m. Preferably, an incubation time at MIT is selected between 10 and 30 seconds at a temperature of 37 to 37.5 and a relative humidity of 90 to ^100/0 .

The test is performed on a human blood-derived buffy coat supernatant sample with a fixed number of leukocytes between 10 and 10 'per sample *. These standard measures are well-established values that aid in excess blood collection do not require the patient «

The buffycoat »sample is added to an antigen solution in the ratio 1: 50 to 1 % 10 · It can be used Antigonlösungen that after one of CASPAR! or obtained by means of a 3 m KOI extraction according to MELTZER * The starting material is human malignant tissue from operations or human brain substance. It is, however, also possible to obtain antigen solution from in vitro grown tumor cells. Such a solution contains between 50 to 200 mg tumor antigen per I ₀ OOO ml of a suitable aqueous solvent "

The test method according to the invention can be applied not only to an unspecific malignant examination but also to an organ-specific examination using antigens obtained from carcinoma tissue of specific organs, thus lymphocytes of patients reacting from the same

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Organ carcinomas are affected, in the described vise. The literature describes that ζ.B, from mammary carcinoma to CASPAR! and FIELD or MELTZER antigen solution give a specific response exclusively to lymphocytes derived from patients with mammacarcinoma.

It is also possible, in the presence of a positive immune response in a general test, i. If a carcinoma is suspected, perform a series of tests with organ-specific antigens to 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, ie the development of the slowing-down effect, can surprisingly be intensified in the movement of the indicator cells in existing tests by adding a solution with a so-called transfer factor to the sample solution. The number of leucocytes from which the added transfer factor amount is obtained is related to the number of leucocytes present in the sample, such as 1 ί 1 to 1 ι 100 »This transfer factor is off

Immunology (see ζ B.M. Rosenthal; The Transfer Factor and Its Therapeutic Use: Switzerland, md. Wschr., Vol. 104, 1974; pages 1501 ... 1506). The transfer factor can be made from normal blood leukocytes. It is a subcellular, dialysable, non-immunogenic leukocyte factor responsible for the T lymphocyte dependent response. There are transfer factor Präpesrate offered (SCHURA, Krefeld), which are produced by fraction in liquid oxygen and subsequent stepwise ultrafractionation. A commercially available unit contains a transfer factor of about 5. 10 leucocytes «The preparation is stabilized with 1% Hunian albumin solution.

It can be seen that with the addition of transfer factor, an enhancement of the immune response can be achieved. The addition of the transfer factor therefore significantly improves the discrimination of the individual sample tests.

Furthermore, the invention relates to a test plate for carrying out the method, which consists of a flat support plate of inert material, for. As glass, there is a 0.5 to 5 mm thick solidified

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Agar layer of 1-20 microns thickness is applied, which has at least two substantially circular punch holes having a diameter between 0.5 and 5 mm (preferably 2.2 to 3 »0 mm). Each punch hole has a capacity of about 5 ··· 20, preferably 8 ul test substance.

It is important that 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 punch hole to the layer.

, Embodiment ...

The invention will be explained by way of examples. In the attached drawing show?

Fig. 1: shows a test plate according to the invention in plan view,

Fig. 2: shows a test plate in sections

example 1

A Geginriung the L eukozyten susp ension

30 ml of human blood are mixed with 300 USP ~ units of ITA

Heparinate heparinized. The heparinized human blood (patient's blood) is underlaid in a plastic tube with 8 ml of 6% by weight dextran solution in physiological saline (molecular weight of dextran: 75,000). Thereafter, the erythrocytes are allowed to sediment at 37 C in a warming cabinet for about 45 min. The leukocyte-rich supernatant (buffycoat) is siphoned off and mixed with the same volume of 0.9% strength by weight NaCl solution, then centrifuged for 15 minutes at an acceleration of 750 g. The pellet depositing in the centrifuge is again washed twice in 10 ml of Hank's solution (pH 7.2) and suspended in a Tc 199 medium (Herst .: Ser ^ a, Heidelberg). During washing and suspending, the cells are whirled up with a pipette until macroscopically no cell clumps are recognizable. The leucocytes are counted after staining with Türkscher solution in the Neubauerkammer and on 2. 1Q cells / μΐ set in Tc 199 medium. On average, 25% of the mononuclear cells (lymphocytes and monocytes) and 75% of granulocytes are obtained for the ax sample. The erythrocyte contamination is less than 1 to 1.

B Preparation of the antigen solution

For the preparation of the antigen solution, the encepha-Ütogene factor (EF) or the basic protein from carcinoma tissue (CaBP) to isolate.

The tumor or brain is treated as quickly as possible after surgical removal under sterile conditions. Connective tissue and healthy tissue are removed from the tumor. Thereafter, the tumor or brain tissue is cut with a pair of sterile scissors (scalpel) into small pieces and further comminuted in the homogenizer in an ice bath. The tumor tissue must not be in this process

heat. 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 for 30 minutes at 2,000 g. The again resuspended by four times the volume of distilled water pellet is freeze-dried. The freeze-dried powder is added in the ratio 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.

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This operation 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 minutes. The supernatant is discarded. With the addition of n / 100 NaCl, the pellet resuspended in five times the volume of distilled water is adjusted to a pH of 3.5. The suspension is gently shaken for 30 minutes, then centrifuged again for 30 minutes at 23,000 g. The supernatant is discarded. Again, the pellet is resuspended in five times the amount of distilled water, which is adjusted to pH 2.6 with n / 100 HCl and allowed to stand for 3 to 18 hours. The pH should not change during this time. The suspension is centrifuged at 23,000 g for 30 minutes. The supernatant is stored, the pellet is washed again in n / 100 NaCl at pH 2.6, centrifuged. The supernatant arising here will be repealed. The latter two supernatants are brought together and then dialyzed against distilled water. This is followed by freeze-drying.

An antigen test solution is made by dissolving

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0/1 mg / ml of freeze-dried substance in distilled water. The basically identical production method also applies to a solution of the basic protein from Tumprgewebe (CaBP).

C Preparation of the Test Plate (See also Figs. 1 and 2)

0.5 g of a special agarose agarose (manufacturer: Behring-Werke, Marburg) are weighed into 50 ml Erlenmeyer flasks. For this purpose, 22.5 ml of sterile bidestillata are added.

Into a second Erlenmeyer flask is added 22.5 ml of the Tc 199 double concentrated medium and 5 ml of human blood derived plasma. In a holder, the agarose is dissolved with gentle circular shaking in a boiling water bath. The Erlenmeyer flask is closed with a foil or a stopper, otherwise a portion of the water would evaporate.

After complete dissolution of the agarose, it is allowed to cool to 47 ° C. in a water bath. In the same bathroom

Then the medium-plasma mixture is preheated and added to the agarose with shaking, so that now 50 ml of a 1% agarose in simple Tc 199 medium with 10% plasma.

With a prewarmed spout each 5 ml of the agar are filled into round tissue culture dishes (Petri dishes) 10 with 50 mm diameter, which are shown in Fig. 1 in plan view. After solidification of the agar, holes 11 with a diameter of 2.5 mm are punched into the agar with the help of a template using a telescopic punching cannula (Behring-Werke, Marburg). When punching out, care must be taken to ensure that the agar layer is not detached from the glass layer in the stamping area so that the capillary layer does not increase and thus the migration of the leukocytes is no longer guaranteed.

The arrangement of the holes 11 results from the Fig. 1. Of course, further Figurationen conceivable. 2 shows an enlarged section through the lower part of the shell 10. It is indicated that the agar layer 1 of the glass layer of the shell 10 by a thin capillary layer. 3

is disconnected. The individual rows of holes are for example intended for: A control (standard) B test suspension with general antigen C test suspension with organ-specific antigen

D Test suspension with organic antigen and transfer factor.

Reaction, incubation and fixation

The following approaches are used for a test:

1. each with 100 μΐ leukocyte suspension to A and 8 ul antigen suspension to B;

2. with 100 μl leukocyte suspension and 8 μΐ η / 100 NaCl solution (for the reference value).

The mixed batches 1 and 2 are incubated for 60 min at 37 ° C in a warming cabinet at 100% humidity. Then, 8 μΐ of leukocyte- and antigen-containing substance or standard substance is introduced from the incubated solution into each punched hole. Depending on the configuration, the evaluation of leukocytic migration and the influence of lymphocyte-produced factors slowing down migration depend on the configuration

the test plate (number of punched holes) 2-5 measurement results and 2-5 reference values available. The cells contained in the suspension, which are introduced into the punched holes at the beginning of the experiment, migrate into the intermediate layer 3 between the agar and the surface of the shell. By incubating the plates at 37. C in the warming cabinet for 18 hours, this walk is maintained over a defined span. The leukocytes do not migrate into the agar, but derive only the nutrients from it. The incubation is carried out at a relative humidity of 100% in a humidity cabinet.

After incubation, plates are overlaid with methanol for 15 minutes followed by formalin solution (37% by weight methanal) for 10 minutes. Subsequently, the agar is carefully peeled off from the shell bottom as skin. The cells migrated in the intermediate layer are now fixed to the surface of the shell. After drying, the cells are stained according to Pappenheim, giving dark purple halos f (migration sites) on the plate surface (see Fig. 1).

E Ausme ssen and 'Determining the results

With a measuring magnifier (Bausch & Lomb) the diameter of the migration halo is measured. In this case, the polymorphonuclear cells rn.it that are isolated in the periphery should be recorded. From each of several measurements, the arithmetic mean is formed.

The quotient of the migration area F ₁ with antigen and the migration area F ₂ without antigen gives a "migration index" MI:

mi = F ₁ Zf ₂ .

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

Row 0 (mm) F (mm ') MI

ß 6,75 F, = 35,8 0,80

A 7.55 F ₂ = 44.75 (1)

Initial examinations in a total of 97 patients were intended to evaluate the effectiveness of the migration inhibition

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Testes in breast carcinoma revealed. Of the breast cancer patients examined, 27 out of 46 showed an inhibition that was greater than 15% (= 58.7%), d. H. the migration index MI is below 0.85. In the 23 examined patients with mastopathy only four out of 23 showed such an inhibition (= 17%), in patients with fibroadenoma one patient of five (= 20%) showed an inhibition. The following table gives a general overview:

diagnosis

Patient number (s)

Migration Index (inhibited) (normal)

Controls 20 Mastopathia cystica 23

Fibroadenoma 5

Mammary cysts 4

other malignant

Diseases 9

Breast Cancer 46

0 (0%) 4 (17%)

1 (20%) 0 (0%)

20 (100%)

19 (83%)

4 (80%)

4 (100%)

3 (33%) 6 (67%) (58.7 %) 19 (41.3%)

Example 2

Use of the transfer factor to enhance the immune response.

It has already been pointed out that by adding a solution with transfer factor (manufacturer: SCHURA, Krefeld) an enhancement of the immune response can be produced. It turns out that when added the factor ΜΪ is more pronounced than in the test example 1.

The following test approaches are made:

1. 100 μΐ leukocyte suspension + 8 μΐ antigen solution

2. 100 μΐ leucocyte suspension + 8 μΐ antigen solution + 20 μΐ solution transfer factor (preparation SCHURA; 1 μΐ corresponds approximately to the transfer factor of 5 × 10 leucocytes)

3. 100 μΐ leukocyte suspension

4. 100 μΐ leukocyte suspension 4 × 20 μΐ transfer factor solution as in batch 2.

Incubation and preparation of the shell are carried out as in Example 1. A shell with four by five punched holes (2.5 mm 0) is produced. One row each with 5 holes is loaded with 10 μΐ of the test mixtures 1 to 4.

Results (each averaged out of five values):

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approach line 0 (mm) F (mm ² ) , MI 1 B 6.90 37.4 0.79 2 C 6.05 28.7 0.61 (including transfer factor) 3 A 7.75 47.2 1.0 standard! 4 D 7.70 46.6 0.99

The low MI of the B / C series indicates a malignancy.

Results with the Migration Inhibition Assay Using the Transfer Factor in the Determination of Gastrointestinal Tumors:

Measurements were carried out with the measuring instructions according to Example 2. There was no significant inhibition in a (healthy) patient control group; the migration index was 1.02 + 0.1. The transfer factor did not influence these results. In the group of patients with colon carcinoma, the migration index was 0.6 5 + 0.2. Of the 15 cases examined, 11 showed a migration index that was less than 0.85. After addition of transfer factor, the migration index was 0.61 + 0.09. Furthermore

In 14 out of 15 MI patients, the transfer factor was less than 0.85, so that a significant improvement in the test result was achieved. In other groups studied no appreciable inhibition could be found.

Example 3

An antigen solution can also be extracted from tumor cells grown in a fermentation culture in vitro with a neutral buffer with repeated disruption of the tissues.

Brain tumor cells are cultured in vitro as follows: care must be taken to use a sterile procedure. The cells are preferably washed in a Hank's balance solution for about 30 seconds. Stirring is to be avoided. All walls and surfaces of the flask, preferably 250 ml flasks of glass, are washed thoroughly. The solution is clarified by the cells by centrifuging in the cold over a period of 10 minutes. The medium is poured into a beaker. »A small amount of buffered proteinase enzyme solution is added

and rinsed quickly to avoid degradation of the cells. In the preferred method, up to two ml of trypsin solution (EDTA) is added in one flask. The trypsin solution is poured off after rinsing 10 sec.

Subsequently, an equal volume of a fresh trypsin solution is added. It is incubated until it can be seen by microscopic observation that the cells separate from the walls of the chamber. This usually takes 5-10 minutes. It is a suitable growth medium, for. B. 50 ml of a solution of a 7-10% solution of the calf fetus serum in 100 ml of F-10 nutrient medium.

25 ml of the fresh medium with the cells are transferred to a new growth chamber (flask) for propagation. Both chambers are placed in a 35 C incubator for approximately 7 days. Following the procedure of this example up to this point, one culture of the artificial tumor cells is divided into two fresh cultures approximately every 7 days. The procedure may be repeated at seven-day intervals. The number of cells growing in vitro doubled

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therefore every seven days.

The medium containing the cells is transferred to a centrifuge tube and centrifuged in the cold for 10 minutes at 3,000 rpm (~ 2,200 g). The medium is discarded. The cells remaining in the growth chamber are scraped off the chamber walls and washed into the centrifuge tubes with a neutral buffer solution. The cells are washed twice with a neutral buffer solution, again centrifuged in the cold at 3,000 rpm (= about 2200 g). The medium is discarded. The washed cells are suspended in 10 ml of neutral phosphate buffer until ready for extraction. Subsequently, the washed cells are treated in the cold for 20 seconds with a sound device to break them. However, the denaturation of the proteins must be avoided as much as possible. After sonication, the cell residues are centrifuged for 30 minutes at 30,000 rpm (= about 220,000 g). The supernatant product is decanted. 10 ml of the buffer solution are used to wash the remaining cell debris. It is again sonicated and centrifuged and the supernatant products are combined. This procedure is repeated once. The combined

supernatant is pre-evaporated to prevent the volume from about 30 ml to about 6-7 ml. A corresponding part is taken for total protein analysis.

Using a liquid column chromatograph and filtration through Millipore filter, the preparation is then freed of impurities. Details of this method can be seen from DT-OS 26 06 257, Example 2. A polypeptide having a molecular weight of about 10,000 can be obtained. An antigen solution is prepared containing approximately 100 mg of the antigen per liter.

The experiments according to Examples 1 and 2 are carried out with the artificially derived antigen. It turns out that the artificially derived antigen produces the same effect as that obtained from body tissue.

Example 4

Use of an organ-specific antigen ^

A Recovery of the leukocyte suspension is carried out as in Example 1 - A.

B Preparation of an organ-specific antigen solution

The most recently obtained tumor tissue of a breast carcinoma is freed from the blood, adipose tissue and connective tissue and cut into small cubes or strips. All subsequent steps are performed at 4C. The GewebsStückchen are comminuted in a threefold volume of a 3-molar potassium chloride solution in approximately pea-sized pieces. The pH of this coarse suspension is adjusted to 7.4. Subsequently, the tissue is comminuted with an Ultraturrax® homogenizer (20,000 Uprn). The homogenate is allowed to stand for 24 hours. It is then centrifuged. The supernatant obtained after centrifugation at 4,000 g for 60 minutes is dialyzed against a 10-fold volume of distilled water for 2 hours and then for a further 24 hours against 50-fold volume of 0.9% NaCl solution. From the dialysate, the proteins are precipitated by addition of 2 molar ammonium sulfate over a period of 1 hour and separated by centrifugation at 4.000 g for 20 minutes. After suspension

The proteins in a 0.9% NaCl solution is dialyzed for 1 hour against 5 times the volume of Agua distillata and a further dialysis against 50 times the volume of 0.3% NaCl. After sterile filtration through a 0.45 milipore micropore filter, the antigen extract is reduced to small vials. filled and stored at -20 ° C. Before use, the protein content of each batch is determined by the biuret method after previous TCA precipitation.

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

Three approaches are produced for the test:

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

2. each with 100 μΐ leukocyte suspension + 8 μΐ antigen suspension according to Example 4 - B,

3. each with 100 μΐ leukocyte suspension + 8 μΐ antigen suspension according to Example 4 - B + 15 μΐ transfer factor solution.

The test plate with a hole diameter of 2.5 mm

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is loaded with each 8 μΐ of the incubated according to Example 1 test substance 1-3. The results, each made up of five values, are:

approach line 7 0 F (mm) MI 1 A 7 70 46.5 1.0 2 C 6 00 38.1 0.82 3 D 10 29.3 0.63.

The result indicates a mammary carcinoma, as the MI is well below 0.8 and the result is even greater when the transfer factor is applied.

Further investigations show that the results of patients, as mentioned in Example 1, change even more significantly. Patients with breast cancer using the method with the help of the transfer factor at over 90%, a migration index, which is less than 0.80. Only in 10% of patients with breast cancer is the migration index greater than 0.85.

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The mobility change of lymphocytes, foreign macrophages and other indicator cells is measured in a capillary test according to S0BORG and BENDIXEN. In this case, a 10 μm thick open capillary is used. The results correspond to those of Examples 1 and 2, using in principle the same approaches.

closing remarks

The test procedure is not applicable for certain clinical pictures. False negative results were found in all so-called leukoses. An explanation for this can not be given at this time, since just leukämisehe cells contain very abundant basic proteins. Also, the cellular immunity in these patients is not so conspicuously disturbed that this result would be explained by a disturbance of the immune system. It is therefore also striking that chronic lymphocytic leukemia, as well as acute lymphatic leukemia, show no reaction, while the lymphosarcomas and lymphogranulomatoses behave like the other malignancies as well. However, prior to chemotherapy indicated

Patients have a significant inhibition. However, after the first cycle of chemotherapy, this inhibition is reversed and turned into a false positive value in the second cycle. Furthermore, there are wrong results in patients with tumor cachexia. Patients with rapidly progressive tumors or patients in tumor cachexia sometimes show incorrect results because of a disturbance of cellular immunity. Attempts to answer this question are still in progress.

False positive results have been found primarily in inflammatory and degenerative neurological disorders, 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 lymphocytes to brain tissue.

However, the wrong results are easily eliminated by a preliminary examination, so that the diseases mentioned do not provide a hindrance to the application of the test.

Claims (1)

AP G 01 Έ / 209 631 54 686 18 Fiction pleasant setti r uch In vitro test method for the diagnosis of malignant tumors, preferably in humans, in which test, the mobility change of cellular blood constituents, which with a. Migration inhi- biting factor (MIF), whereby the MIF is excreted by lymphocytes which are sensitized to tumor tissue, characterized in that, as cell-shaped blood components, the lymphocytes to be examined, as part of a leukocyte suspension, undergo a migration reaction which is known per se. Inhibition test be submitted, in the course of which the lymphocytes are brought into contact with an MTF to the stimulating antigen and then incubated, and after a fixed period of time, the leukocyte migration is inhibited and fixed? Test method according to item 1 _S d by _a that the Wanderungsgeschwindigkeitcer leukocytes in a capillary intermediate layer of 1-20 to Dickej. which is at least partially filled with liquid, is measured ₀ Test method according to item 2, characterized in that on the one hand the intermediate layer is covered by an inert AP G 01 π / 209 54 686 18. th medium (for example, _E glass) and the other side by an enriched 'Nutrient agar layer is * Test method according to one of the items 1 to 3 »characterized in that the test is carried out with a sample of human blood-derived leucocyte supernatant (" buffy coat ") with a fixed number of leucocytes between 10 ^ and 10 'per sample." 5c test method according to item 4, characterized in that the sample is added to an antigen solution according to CASPAEX and FISLD or a 3 m KCl extract from tumor tissue according to MELTZER in the ratio (volume sample to antigen solution) of 1 % 60 to 1 s 10 » Test method according to item 5s, characterized in that an organ-specific antigen solution is added to the sample. 7 * Test method according to item 4, characterized in that: an antigen solution obtained from in vitro cultivated tumor cells is added to the sample, v; el ehe between 50 to 200 mg tumor antigen per 1,000 ml of a suitable aqueous solvent contained 8c. Test method according to any one of items ¹ to 7 A- »characterized in that _f the sample in addition to the antigen solution additional transfer -.? Actuator solution 'is added. AP G 01 N / 209 631 54 686 18 9 _β test method according to item 8, characterized _s that the number of leukocytes, from which the added transfer factor amount is obtained, to the number of leukocytes present in the sample, such as 1; 1 to 1% 100 'behaving , Test method according to one of the preceding points, characterized in that the incubation time in the migration inhibition test is between 10 and 30 hours at a temperature of 35 to 37> 5 ^° C at a relative humidity of 90 to 100%. " 11 «Test plate for carrying out the method according to one of the items 1 to 10, characterized by a flat carrier plate or tray (10) of inert material (z, B, glass), to which a 2 - 5 ™ thick, solidified agar layer is applied, wherein between the support plate or shell (10) and. the agar layer has a capillary migration layer of 1-20 μm thickness and the agar layer has at least two substantially circular. punch holes (11) with a diameter between 0.5 and 5 mm, preferably 2.5 mm. " 12® test plate.According to item 11, characterized in that the punched holes are cut in such a way that the uniformity of the capillary migration layer is also present in the region of the transition from the punched hole to the capillary layer. « For this purpose / f. Page drawing