DE2755363B2 - Test method for diagnosing malignancies - Google Patents

Description

The invention relates to an in vitro test method for

.'ο Diagnosis of malignant tumors, preferably at People, in which test the change in mobility of cellular blood components that are associated with a Migration Inhibition Factor (MIF) in contact is determined, the MIF being determined by lymphocytes

r> excreted which are sensitized to tumor tissue. Such a MIF will not excreted if the lymphocytes come from patients who do not have a malignant tumor (malignancy), because these lymphocytes are not against that

jo tumor antigens used are sensitized and therefore do not release MIF on contact with tumor antigen.

From the literature (Douwes, F. R. et al .; Verh. Deutsche Ges. Inn. Med., Volume 82, J. F. Bergmann-Ver

r> lag, Munich 1976; there further evidence) is known that with the help of the so-called electrophoresis mobility test (EMT) early detection of malignant diseases is possible. The principle of the EMT is based on the fact that the migration speed (mobility) of certain bodies (macrophages, Granulocytes, other blood cells) only changes when they are sensitized with lymphokines Lymphocytes come into contact. It will

• r. provided that lymphocytes that are sensitized to a certain antigen (so-called T lymphocytes), release a number of soluble factors called lymphokines. An important one among these Factors is the Migration Inhibition Factor (MIF).

^r , o It influences the ability of blood cells to migrate, in particular macrophages and granulocytes, and ensures that they are locked in place and accumulate at the site of the presence of the antigen which triggers the sensitization. In vivo means the destructive

v ") possibility of the tumor.

Lymphocytes from malignant patients react specifically by releasing the so-called soluble factors when they come into contact with a basic protein that is made up of brain matter

ho on the one hand or from a carcinoma tissue on the other is taken. The basic proteins that cause the specific reactions of the lymphocytes, encephalitogenic factor (EF) and basic protein from carcinoma tissue (CaBP)

iv> called. The extraction takes place according to known methods Method (Caspary, E.A. et al., Brit. Med. 1971, pp. 613-617 and other publications; see Douwes, F. R. a. a. O.).

The results of blood tests with the EMT described in the literature show that with the help of the test an almost 100% differentiation of the samples can be made in terms of whether they are a Organism with malignant (tumor) diseases or a healthy organism or an organism with non-malignant diseases. All examined patients with (malignant) Tumors showed a positive lymphocyte response; H. it became a fakior from the lymphocytes released, which leads to a measurable and characteristic change in the mobility of indicator cells (cf.L Douwes, F. R, op. cit.). Check-ups Patients with non-malignancies did not show such a lymphocyte response. False results could be traced back to certain causes. Accordingly, the EMT may be the most sensitive at the moment Cancer test to be viewed.

A disadvantage of using the test is that it is relatively complicated to master instrument to be used, namely the cytopherometer, is required. That is the speed of migration Observing and measuring personnel must be trained and monitored very carefully. Because of the waning attention only a limited amount of work is possible. The application of the EMT is therefore limited to specialized institutes. The evaluation of the test takes place during the movement of the blood cells, so dynamic. It is not possible to "stop" the experiment, so there is no re-measurement perform.

The object of the invention is to provide a test method for diagnosing malignancies, that as general and unspecific as possible with regard to the type of malignancy with a high degree of accuracy The presence of malignancies is signaled, but in principle it is set up or used in every doctor's laboratory. can be carried out and is particularly suitable for mass screening in cancer screening. The result of the test should be readily measurable. The test should be statistically evaluable and without Intermediate logging can be archived.

This object is achieved in a test method of the type mentioned in that as cellular blood components the lymphocytes to be examined as part of a leukocyte suspension subjected to a migration inhibition test known per se, in the course of which the lymphocytes brought into contact with an antigen that stimulates the formation of MIF and then incubated, and after a fixed period of time, the migration of leukocytes is inhibited and fixed.

There are various migration inhibition is tests known, which are basically suitable for the investigation according to the task. Is known under among other things the so-called capillary technique according to S0BORG and BENDIXEN. To realize the idea of the invention it is necessary to increase the mobility of the indicator cells by »freezing« one after a certain time to measure the migration status reached.

According to the invention, the spread or change in mobility of the leukocytes within a very fine, approximately ΙΟμηι thick »open« Capillary layer measured. A test arrangement that consists of a Two-layer element consists, namely of a carrier made of an inert medium, e.g. B. Glass, and one Agar, which is enriched with nutrients for blood cells. A particularly suitable agar is one under the Name agarose: commercially available agar, which is manufactured by the company Behring-Werke, Marburg.

The intermediate layer between agar and carrier layer is preferably partially filled with liquid has a thickness of about 1-20 μm. Preferably an incubation time for MIT between 10 and 30 hours is chosen.

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

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 obtained according to a method specified by CASPARY and FIELD or obtained with the aid of a 3 m KCI extraction according to MELTZER. As a starting material serve human malignant tissue from operations or human brain matter. However, it is it is also possible to obtain antigen solution from tumor cells 7u grown in vitro.

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 affected by the same organ carcinoma in the manner described. In the literature will described that z. B. obtained from breast carcinoma 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. H. at 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 Surprisingly, movement of the indicator cells in the present tests can be increased by that a solution with a so-called transfer factor is added to the sample solution. This factor is from the Immunology known (see e.g. M. Rosenthal: The transfer factor and its therapeutic application; Switzerland, md. Wschr, Vol. 104, 1974; Pp. 1501 ... 1506). Of the Transfer factor can be made from normal blood leukocytes. It represents a subcellular, dialyzable, non-immunogenic leukocyte factor that is responsible for the T-lymphocyte-dependent reaction responsible for. Transfer factor preparations are available (SCHURA, Krefeld) that use fractionation in liquid oxygen and subsequent gradual clock fractionation. One commercially available unit contains transfer factor of approx. 5 · 10 '"leukocytes. The preparation is 10% higher Stabilized human albumin solution.

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

the addition of the transfer factor therefore makes the discrimination of the individual sample tests even more important improved.

The invention further relates to a test plate for carrying out the method, which from a flat support plate or tray made of inert material (e.g. glass) on which a 2-5 mm thick, solidified agar layer is applied, being between the carrier plate or tray and the Agar layer is a capillary migration layer of 1-20 μm thickness and the agar layer is 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 5-20, 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 included:

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 Na heparinate. The heparinized human blood (patient's blood) is placed under a layer of 8 ml of 6% by weight dextran solution in physiological saline (molecular weight of the dextran: 75,000) in a plastic tube. The erythrocytes are then allowed to sediment at 37 ° C. in a heating cabinet for about 45 minutes. 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 min at an acceleration of 750 g. The pellet which is deposited in the centrifuge is again twice in 10 m! Hank's solution (pH 7.2) and suspended in a Tc 199 medium (Manuf .: Serva, Heidelberg). When washing and suspending the cells are swirled up with a pipette 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 encephalitogene is used to prepare the antigen solution To isolate factor (EF) or the basic protein from 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 afterwards the tumor or brain tissue is cut into small pieces with sterile scissors (scalpel) and im Homogenizer further crushed in an ice bath. The tumor tissue must not be removed during this process heat a The homogenate is suspended in four times the volume of distilled water and then centrifuged at 23,000 g. The resulting pellet wire again in four times the volume of 0.9% NaCl-Lo solution suspended, homogenized and centrifuged for 30 min at 23,000 j. That in turn in four times the volume

· - Pellet resuspended in distilled water is freeze-dried. The freeze-dried powder is in Ratio 1:10 with a chloroform / methanol Ge mixed (2: 1) added and stirred for 30 min. The Gemiscl is centrifuged at 600 g for 10 min. The Überstanc

κι is discarded.

This operation is repeated twice. There; Pellet is air dried. The dry pellet is in Resuspended five times the volume of a 5% NaCl solution with water and centrifuged at 23,000 g for 30 min

ι · ■> yaws. The supernatant is discarded. Under encore before n / 100 NaCl, the pellet is again resuspended in five times the volume of distilled water adjusted to a pH of 3.5. The suspension is shaken gently for 30 minutes, then centrifuged again for 30 minutes

2 (i 23,000 g. The supernatant is discarded. Again i the pellet is resuspended in five times the amount of distilled water, which is mixed with n / 100 HCl on a pH is adjusted to 2.6 and left to stand for 3 to 18 hours. The pH value is allowed to persist

2) do not change this time. The suspension will be Centrifuged 23,000 g for 30 min. The supernatant is stored, the pellet is again in n / 100 NaCl at pH 2.6 washed, centrifuged The supernatant produced here is saved. The latter two

in supernatants are brought together and danr dialyzed against distilled water. This is followed by freeze-drying.

An antigen test solution is made by dissolving 0.1 mg / ml of the freeze-dried substance in distil

j-, get lated water. That is basically the same The manufacturing process also applies to a solution of 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; manuf ler Behring-Werke, Marburg) are in 50 ml alder Meyer flask weighed in. To this end, 22.5 m of sterile double-distilled water are added.

• r, 22.5 m. Into a second Erlenmeyer flask des double concentrated Tc 199 medium and 5 ml au; Given plasma obtained from human blood. In one In the holder, the agarose is dissolved in a boiling water bath while shaking gently in a circular motion. The alder

-, egg Meyer 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. In the same bathroom wire then the medium-plasma mixture is preheated and added to the agarose while shaking so that now 50 ml of a 1% agarose in plain Tc 199 medi to be present with 10% plasma.

5 ml of the agar are poured into round tissue culture dishes using a preheated discharge pipette (Petri dishes) 10 filled with 50 mm diameter, which in F i g. 1 are shown in plan view. After the agar has solidified, a telescopic punch cannula (Behring-Werke, Marburg) with the aid of a template, holes 11 with a diameter of 2 ^ mm in the agar punched When punching out, care must be taken that the agar layer in the punched area does not come off the Glass layer is peeled off so that the capillary layer

not enlarged and so the migration of the leukocytes is no longer guaranteed.

The arrangement of the holes 11 is shown in FIG. 1. Further figurations are of course possible 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 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 for a test:

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

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

The mixed batches 1 and 2 are for 60 min at 37 ° C in a heating cabinet with 100% humidity incubated. Then 8 μl leukocytes and antigen-containing from the incubated solution are put into each punch hole Substance or standard substance filled in. For the evaluation of the leukocyte migration and the influence by the factors produced by the lymphocytes, which slow down the migration, are dependent on each other after configuration of the test plate (number of punched holes) 2–5 measurement results as well as 2–5 reference values for Disposal. The cells contained in the suspension that were 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 a heating cabinet for 18 hours, this hike over a defined span maintain. The leukocytes do not migrate into the agar, but only obtain the from it NLhrients. The incubation is carried out at a relative humidity of 90-100% in a humidity cabinet carried out.

After incubation, the plates are washed with methanol for 15 minutes and then with formalin solution for 10 minutes (37 wt .-% methanal) overlaid. Then the agar is carefully removed as a skin Dish bottom peeled off. The cells that have migrated in the intermediate layer are now on the surface of the Fixed shell. After drying, the cells are stained according to Pappenheim, turning dark purple Halos 4 (wandering courtyards) on the plate surface result (see Fig. 1).

E measuring and determining the results

The diameter of the Vanderungshalo is measured with a magnifying glass (Bausch & Lomb). Included the isolated polymorphonuclear cells in the periphery must also be recorded. From each the arithmetic mean is calculated for several measurements.

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

MI = F ₁ ZF 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:

0 (mm)

6.75
7.55

F, = 35.8
F ₂ = 44.75

Initial studies on a total of 97 patients were to determine the effectiveness of the migration inhibition test in breast cancer. 27 of 46 examined patients with breast cancer show an inhibition that is greater than 15% (= 58.7%), d. H. the migration index MI is below 0.85. In the 23 examined patients with mastopathy only show four of 23 showed such an inhibition (= 17%), in patients with fibroadenoma one patient of five (= 20%) one inhibition. The following table gives a general overview:

diagnosis Patient number Migration index (normal) (n) (inhibited) 20 (100%) Controls 20th 0 (0%) 19 (83%) Mastopathia cystica 23 4 (17%) 4 (80%) Fibroadenoma 5 1 (20%) 4 (100%) Breast cysts 4th 0 (0%) 6 (670/0) Other malignancies 9 3 (33%) 19 (41.3%) Breast cancer 46 27 (58.7%)

Example 2

Use of the transfer _factor to enhance the immune response

It has already been pointed out that by adding a solution with a transfer factor (manufacturer: SCHURA, Krefeld) an enhancement of the immune response can be induced. It turns out that with Addition of factor MI is more pronounced than compared to test example 1.

The following test approaches are made:

1. 100 μΐ leukocyte suspension + 8 μΐ antigen solution

2. 100 μΐ leukocyte suspension + 8 μΐ antigen solution

+ 20 μΐ solution transfer factor (SCHURA preparation; 1 μΐ 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.

Incubation and preparation of the dish are carried out as in Example 1. It becomes a dish with four by five Punched holes (2.5 mm 0) made. Each row with 5 holes is loaded with 10 μl of the test batches 1 to 4.

Results (each averaged from five values):

approach line 0 (mm) FW I 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 lower Ml of rows B / C indicates eir.in 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 2. In a (healthy) patient control group there was no significant inhibition, the migration index was 1.02 ± 0.1. The transfer factor did not affect these results. In the group of patients suffering from colon carcinoma was the Migration index at 0.65 ± 0.2. Of the> 5 examined 11 cases showed a migration index that was less than 0.85. After adding transfer factor the migration index was 0.61 ± 0.09. In addition, 14 of 15 patients had MI after the addition transfer factor below 0.85, so that here too a significant improvement in the test result had been achieved. No significant inhibition could be found in other groups examined.

Example 3

Use of an organ-specific antigen
A The leukocyte suspension is obtained

As in uciSpici ι - / Λ

B Preparation of an organ-specific antigen solution

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. All subsequent 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. The tissue is then comminuted with an Ultraturrax® homogenizer (20,000 rpm). 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. Ammonium sulfate is added to the dialysate over a period of time The proteins are precipitated within 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 distilled water and another dialysis against 50 times the volume of 0.9 ° % NaCl. After sterile filtration through a 0.45 Millipore Micropore filter the antigen extract is filled into small vials and stored at -20 ^0C. Before use, the protein content of each batch is determined using the biuret method after previous TCA precipitation.

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

Three approaches are made for the test:

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

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

3. each with 100 μl leukocyte suspension + 8 μl '' Antigen suspension according to Example 4-B + 15 μΙ

Transfer factor solution.

The test plate with a hole diameter of 2.5 mm is incubated with 8 μΙ each according to Example 1 2 (i test substance 1 - 3 loaded. The results, each from five values determined are:

approach

line

F (mm-)

Ml

1 A. 7.70 46.5 2 C. 7.00 38.1 3 D. 6.10 29.3

1.0

0.82

0.63

κι The result suggests breast cancer, da the MI is well below 0.85 and the result is reinforced when the transfer factor is applied.

Further investigations show that the results of patients, as mentioned in Example 1, are still

r, change much more significantly. Patients with Breast cancer show when using the method with the help of the transfer factor in over 90% a migration index that is less than 0.80. This is only found in 10% of patients with breast cancer

κι migration index greater than 0.85.

Example 4

The change in mobility of lymphocytes, foreign macrophages and other indicator cells in 4-, a capillary test according to S0BORG and BENDIXEN. It is here a 10 μίτι thick open Capillary used. The results correspond to those of Examples 1 and 2, with 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

· -, · -, cannot be given for this 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.

M) It is therefore also noticeable that the chronic lymphatic Leukemia, as well as acute lymphoblastic leukemia, show no response, while the lymphosarcomas and the lymphogranulomatoses behave like the other malignancies. Before chemotherapy

However, patients showed a clear inhibition of bs pie on. After the first chemotherapy cycle, however, this inhibition is lifted and im second cycle to a false positive value

11 12

transformed. Furthermore, false results are found diseases, for example multiple sclerosis,

in patients with tumor cachexia. Patients with Rapidly The explanation for this has been given by Caspary and Field in

progressively growing tumors or patients in which there is a cross reaction between the EF and the

Tumor cachexia sometimes show incorrect results because the lymphocytes are sensitized with brain tissue

there is a disorder of cellular immunity. ■> seen.

Attempts to answer this question are still in the making. However, the wrong results are easily covered by one

In progress. Preliminary examination to be eliminated so that the said

False positive results were primarily not an obstacle to the application of diseases.

provide inflammatory and degenerative neurological grounding of the test.

For this 1 blue drawing

Claims (12)

Patent claims: 1. In vitro test methods for the diagnosis of malignant tumors, preferably in humans, in which test the change in mobility of cellular blood components with a Migration Inhibition Factor (MIF) contact is determined, the MIF of Lymphocytes are excreted which are sensitized to tumor tissue, characterized in that that as cellular blood components the lymphocytes to be examined as part of a leukocyte suspension in itself known migration inhibition test are subjected, in the course of which the lymphocytes with brought into contact with an antigen which stimulates the formation of MIF and then incubated and after a fixed period of time the migration of leukocytes is inhibited and fixed. 2. Test method according to claim 1, characterized in that the migration speed of leukocytes in a capillary intermediate layer of 1–20μΐη thickness, which is at least is partially filled with liquid, is measured. 3. Test method according to claim 2, characterized in that the intermediate layer on the one hand from an inert medium (e.g. glass) and on the other hand from one enriched with nutrients Agar layer is limited. 4. Test method according to one of claims 1 to 3, characterized in that the test is carried out with a sample with ieukocyte-rich supernatant ("buffycoat") obtained from human blood with a fixed number of leukocytes between 10 ⁴ and 10 ⁷ per sample. 5. Test method according to claim 4, characterized in that the sample is an antigen solution according to CASPARY and FIELD or a 3-m-KCI - & [- tract from tumor tissue according to MELTZER im Ratio (volume sample to antigen solution) of 1:50 to 1:10 is added. 6. Test method according to claim 5, characterized in that the sample is an organ-specific Antigen solution is added. 7. Test method according to claim 4, characterized in that the sample is one grown from in vitro Antigen solution obtained from tumor cells is added, which is between 50 to 200 mg Contains tumor antigen per 1000 ml of a suitable aqueous solvent. 8. Test method according to one of claims 4 to 7, characterized in that the sample in addition to the Antigen solution additional transfer factor solution is added. 9. Test method according to claim 8, characterized in that the number of leukocytes from which the added amount of transfer factor is recovered depends on the number of leukocytes present in the Sample are present as 1: 1 to 1: 100 behaves. 10. Test method according to one of the preceding claims, characterized in that the incubation time in the migration inhibition test between 10 and 30 hours at a temperature of 35 to 37.5 ° C with a relative humidity of 90-100%. 11. Test plate for performing the method according to one of claims 1 to 10, characterized by a flat support plate or tray (10) made of inert material (e.g. glass) onto which a 2-5 mm thick, solidified agar layer is applied, with between the support plate or tray (10) and the agar layer has a capillary migration layer 1–20μΐη thick and the agar layer at least two substantially circular punched holes (11) with a diameter between 0.5 and 5 mm, preferably 2.5 mm 12 test plate according to claim 11, characterized in that that the punched holes are cut so that the uniformity of the capillary migration layer also in the area of the transition from Punch hole is given to the capillary layer